GANP protein

ABSTRACT

The object of the present invention is to provide a novel protein having a kinase activity and a gene encoding said protein. According to the present invention, there is provided a GANP protein which is represented by the amino acid sequence shown in SEQ ID No. 1 or 3 of the sequence listing, and is involved in the signal conversion of abnormal B cell differentiation in an autoimmune state, and has a kinase activity, and a polynucleotide which encodes said protein.

This application is a Divisional of U.S. application Ser. No. 09/914,272filed Dec. 5, 2001, now U.S. Pat. No. 6,673,913 issued Jan. 06, 2004,which is a 371 of PCT/JP99/04634 filed Aug. 27, 1999.

TECHNICAL FIELD

The present invention relates to a novel protein having a kinaseactivity and a gene encoding said protein.

BACKGROUND ART

Antigen binding to the membrane lgR initiates the activation andmaturation of the antigen-specific B cells in the peripheral lymphoidorgans (Rajewsky, Nature (Lond.)., 381:751-758, 1996; Sakaguchi et al.,Adv. Immunol. 54:337-392, 1993). B cells enter the outer periarteriallymphoid sheath (PALS) (Rajewsky, Nature (Lond.)., 381:751-758, 1996)and initiate costimulus-dependent interactions with specific Th cellsand interdigitating dendritic cells within 48 h after immunization(MacLennan, Annu. Rev. Immunol. 12:117-139, 1994; Liu et al., Immunol.Rev. 156:111-126, 1997). Antigen-driven B cells proliferate in the outerPALS and then undergo further activation in the lymphoid follicles toestablish the germinal center (herein sometimes abbreviated as GC) (Hanet al., J. Immunol. 155:556-567, 1995; Jacob et al., J. Exp. Med.176:679-687, 1992; Kelsoe, Immunity 4:107-111, 1996). Such B cellsmature into large slg⁻ centroblasts that rapidly move through the cellcycle to form the dark zone and further mature into centrocytes thatexpress a unique surface character of PNA⁺B220^(+slgM) ⁺slgD⁻CD38⁻ inthe light zone of the GC (Kosco-Vilbois et al., 1997. Immunol. Today18:225-230, 1997; Kelsoe, Immunol. Today 16:324-326, 1995; Oliver etal., J. Immunol. 158:1108-1115, 1997).

Centrocytes presumably undergo the processes of either apoptosis oraffinity maturation of immunoglobulin V regions and the change processof class switching toward the IgG class antigen. Some centrocytessurvive for a longer period in the lymphoid compartment as memory Bcells. The other centrocytes probably migrate to the marginal zone ofthe GC and receive further antigenic stimulation and costimulatorysignals through B cell activation molecules, such as CD40 and CD38, andreceptors for various B cell stimulatory cytokines (Gray et al., J. Exp.Med., 180:141-155, 1994; Foy et al., J. Exp. Med., 180:157-163, 1994).Antigen-specific B cells further stimulated in this area probablymigrate into the interstitial region of the spleen (called red pulp),where various kinds of other immune-competent cells may interact withantigen-driven B cells. Histochemical analysis in several autoimmunemice identified unique antibody-producing cells in this area whichappear as plasma cells or aberrant plasma cells called Mott cells(Tarlinton et al., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J.Immunol., 158:992-997, 1997).

Autoimmunity is a phenomenon in which the impairment of self/nonselfdiscrimination occurs frequently in the antigen-specific lymphocytes(Theofilopoulos, Immunol. Today, 16:90-98, 1995). The immune systems ofvarious autoimmune diseases show the combinatory mechanism involving Tcells and B cells (Theofilopoulos et al., Adv. Immunol., 37:269-290,1985; Okamoto et al., J. Exp. Med. 175:71-79, 1992; Reininger et al., J.Exp. Med., 184:853-861, 1996; Theofilopoulos, et al., Immunol. Rev.55:179-216, 1981; Watanabe-Fukunaga et al., Nature (Lond.).,356:314-317, 1992; Takahashi et al., Cell, 76:969-976, 1994; Shlomchicket al., Nature (Lond.). 328:805-811, 1987).

NZB and NZW are the strains characterized by multiple genetic factorsgenerating the severe autoimmune state of SLE as (NZB×NZW)F₁ mice(Theofilopoulos et al., Adv. Immunol., 37:269-290, 1985; Okamoto et al.,J. Exp. Med., 175:71-79, 1992; Reininger et al., J. Exp. Med.,184:853-861, 1996; Theofilopoulos et al., Immunol. Rev., 55:179-216,1981). NZB mice spontaneously generate the state of autoimmunity withthe anti-red blood cell antibody that causes an autoimmune hemolyticanemia (Okamoto et al., J. Exp. Med., 175:71-79, 1992). NZW mice show aninsidious autoimmune phenomenon (Reininger et al., J. Exp. Med.184:853-861, 1996). The SLE state of (NZB×NZW)F₁ mice is apparentlycaused by multiple genetic factors associated with T and B cells(Theofilopoulos et al., Immunol. Rev., 55:179-216, 1981). NZB mice showan apparent abnormality of B cells, but the molecular mechanism of theabnormal B cell activation in NZB mice remains to be elucidated.

DISCLOSURE OF THE INVENTION

To address the issue of which molecules are involved in such maturationof B cells, the present inventors prepared monoclonal antibodies againstintracellular components of a murine B cell line WEHI-231, which has theNZB genetic background. A monoclonal antibody named 29-15 recognizes adifferentiation antigen whose expression is augmented in GC-B cells ofperipheral lymphoid organs. With the 29-15 monoclonal antibody, thepresent inventors studied the expression of the antigen in peripherallymphoid organs, which characterized the molecule as a differentiationantigen upregulated in the light zone of the GC from hyperimmunizedmice. In the spleen of NZB mice, IgM-producing plasma cells with highexpression of the GANP antigen appear before the onset of autoimmunity,which would suggest that this is an important molecular event forunderstanding the peripheral immune response and autoimmunity with autoantibodies.

The present inventors have studied to identify the above-mentionedantigen whose expression is selectively increased in centrocytes ofgerminal center, and confirmed by in situ RNA hybridization using anisolated cDNA probe (ganp probe) that the expression of ganp mRNA isincreased in the area stained with 29-15 monoclonal antibody. It wasalso confirmed that the gene product, GANP protein, is a protein of 210kD which is localized in cytoplasma and nucleus, and is structurallysimilar with a transcription regulating factor in yeasts, SAC3. When Bcells are activated with anti-IgM antibody and anti-CD40 antibody, theamount of kinase which binds to GANP protein increased. These resultssuggest that GANP protein may be involved in a signal conversion ofabnormal B cell differentiation in certain autoimmune state. The presentinvention has been completed on the basis of these findings.

Thus, the present invention provides a GANP protein represented by theamino acid sequence shown in SEQ ID No.1 or No.3 of the sequencelisting. According to the present invention, there is provided a GANPmutant protein which is consisted of the amino acid sequence wherein oneor more amino acids are deleted, one or more amino acids are substitutedwith other amino acid(s), and/or one or more other amino acids are addedin the amino acid sequences shown in SEQ ID No.1 or No.3 of the sequencelisting, and has a kinase activity similar with that of GANP protein.According to the present invention, there is provided a polypeptidewhich contains, as a partial sequence, a full length amino acid sequenceof the aforementioned GANP protein or the aforementioned GANP mutantprotein.

According to another aspect, the present invention provides apolynucleotide which encodes the aforementioned GANP protein or GANPmutant protein. The typical polynucleotide is DNA encoding GANP proteinderived from mammal, and the DNA of mammal gene is preferred among them.Examples of most preferred polynucleotide are represented by the basesequences shown in SEQ ID No. 2 (DNA sequence encoding GANP protein frommouse) or SEQ ID No. 4 (DNA sequence encoding GANP protein from human)of the sequence listing.

Further, according to the present invention, there is provided anantisense polynucleotide which is composed of the base sequence of anantisense chain of the aforementioned polynucleotide, or derivatives ofsaid antisense polynucleotide. Furthermore, according to the presentinvention, there is provided a polynucleotide or antisensepolynucleotide of continuous 12 or more bases which is a partialsequence of the aforementioned polynucleotide or the aforementionedantisense polynucleotide, and a chemically modified polynucleotide orantisense polynucleotide of the aforementioned polynucleotide or theaforementioned antisense polynucleotide.

According to further another aspect, the present invention provides amethod for obtaining DNA of the base sequence shown in SEQ ID No. 2 orNo. 4 of the sequence listing or DNA which is the homologue from othermammal, wherein the aforementioned polynucleotide or antisensepolynucleotide is used as a probe, and cDNA which hybridizes to theprobe is obtained from mammal cDNA library. The length of the cDNA isalmost the same as that of GANP gene, and the protein encoded by it hasapproximately 210 kDa. Further, according to the present invention,there is provided cDNA obtained by the aforementioned method and GANPprotein encoded by it.

According to further another aspect of the present invention, there isprovided an antibody which recognizes GANP protein or GANP mutantprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing detection of 29-15⁺ cells in the PP ofnormal mice. The immunohistochemical analysis was carried out on PP withthe 29-15 mAb and ALP-anti-rat 1 g antibody. Positive cells appear inthe central area with Vector Blue ALP substrate, and the strong signalin the surrounding area is in intestinal villi containing nonspecificendogenous ALP activity. For two-color staining, the sections arefurther stained either with biotin-anti-B220 mAb or biotin-anti-IgD mAbfollowed by HRP-streptavidin in combination with DAB.

FIG. 2 is a photograph showing appearance of 29-15⁺ cells in the GC areaof SRBC-immunized mice. Normal BALB/c mice were injected four times withSRBC during 12 days and the spleen sections were stained withhematoxylin or studied by immunohistochemistry as in FIG. 1. Thesections of normal and SRBC-immunized BALB/c mice are parallel, whencompared after staining with the 29-15 mAb.

FIG. 3 is a photograph showing appearance of 29-15⁺ cells in the GC areaof SRBC-immunized mice. The sections of the GC area are stained withPNA, anti-BrdU, and the 29-15 mAb in combination with the individualcolors as described in the Materials and Methods. Upper photograph showshematoxylin staining of the GC area (GC) and the central artery (CA).Middle photograph shows three-color staining, indicating the 29-15⁺PNA⁺cells. Lower panel shows a schema of 29-15⁺PNA⁺ cells.

FIG. 4 is a photograph showing expression of the GANP^(dense+) cells inthe red pulp area of autoimmune-prone mice. Sections were prepared fromthe spleens of nonimmunized mice of BALB/c, NOD, NZB, (NZB X NZW)F₁,BXSB, and MRL/lpr. All mice were used 6-8 weeks after birth. TheGANP^(dense+) cells stained with the 29-15 mAb appear in the red pulparea of NZB, (NZB X NZW)F₁, MRL/lpr, and BXSB strains.

FIG. 5 is a photograph showing expression of the GANP^(dense+) cells inthe red pulp area of autoimmune-prone mice. Sections of the LN ofpopliteal regions were stained with the 29-15 mAb. The GANP^(dense+)cells appear in peripheral LN of older NZB mice (10 month old) andMRL/lpr mice (8 week old).

FIG. 6 is a photograph showing characterization of the GANP^(dense+)cells in the autoimmune-prone mice. Sections were prepared with thespleen of nonimmunized NZB mice (8 week old). Immunohistochemicalanalysis was performed with the 29-15 mAb in combination with one of thefollowing reagents: anti-B220, PNA.

FIG. 7 is a photograph showing characterization of the GANP^(dense+)cells in the autoimmune-prone mice. Sections were prepared with thespleen of nonimmunized NZB mice (8 week old). Immunohistochemicalanalysis was performed with the 29-15 mAb in combination with one of thefollowing reagents: anti-lgM, anti-Syndecan-1, or anti-BrdU mAb.

FIG. 8 is a photograph showing Mott cells that appear in NZB mice by PASstaining.

FIG. 9 is a diagram showing a deduced amino acid sequence of mouse GANPprotein in one character notation SEQ ID No: 1.

FIG. 10 is a diagram showing a structure of the GANP protein. In thefigure, S/T rich region: serine/threonine rich region, SAC3 homologyregion; SAC3 homology region, nuclear localizing signal: nuclearlocalizing signal. Four LXXLL motifs are present.

FIG. 11 is a photograph showing a result of in situ RNA hybridization ofthe ganp gene. Sections of spleens from SRBC-immunized, nonimmunizedBALB/c, and NZB mice were hybridized with the ganp anti-sense probe. Inthe figure, the white pulp area (WP), red pulp area (RP), and GC area(GC) are indicated The GANP^(dense+) cells were recognized in the redpulp of NZB mice.

FIG. 12 is a diagram showing the results of the analysis by Westernblotting after immunoprecipitation of GANP protein. The GANP protein wasdetected as a 210-kD protein expressed in cytoplasmic and nuclearfractions of WEHI-231 cells.

FIG. 13 is a diagram showing the results where spleen B cells fromnormal BALB/c mice were stimulated with F(ab′)₂ of goat anti-lgM Ab (10μg/ml) and anti-CD40 mAb (10 μg/ml) for 48 hour and stained with theanti-GANP mAb.

FIG. 14 is a diagram showing the results where in vitro kinase reactionwas carried out with the anti-GANP immunoprecipitates in the presence of[γ-³²P]-ATP for 10 minutes. Phosphorylation on the proteins weredetected by the autoradiography after SDS-PAGE separation.Phosphorylation of the GANP is indicated with an arrow (Figure A), andphosphoamino acid analysis of phosphorylated GANP protein is also shown(Figure B).

FIG. 15 is a diagram of the structure of the mouse GANP protein. In thefigure, the homologous region to SAC3 and Map80, nuclear localizationsequences (NLSs), and coiled-coil regions are indicated. Four LXXLLmotifs are indicated by black.

FIG. 16 shows a result of RT-PCR assay. The upregulation of gnap mRNA inanti-μ- and anti-CD40-stimulated B calls in vitro is shown. HPRT wasused as a control to confirm the amount of each template.

FIG. 17 shows a result of in vitro kinase reaction. The call lysate wasprepared from unstimulated (left) or stimulated (right) cells andsubjected to anti-GNAP immunoprecipitation. In vitro kinase reaction wascarried out with the anti-GNAP (42-23) immunoprecipitates in thepresence of [γ-³²P]-ATP for 10 minutes. Phosphorylation on the proteinswas detected by the autoradiography after SDS-PAGE separation. An arrowindicates the position of phosphorylated GNAP.

FIG. 18 is a scheme showing a physical association between GNAP andMCM3. The cell lysate from WEHI-231 was immunoprecipitated withanti-GST, anti-GNAP (42-23), or anti-MCM3 Ab. After separation bySDS-PAGE, the proteins were electrophoretically transferred to amembrane and probed with anti-MCM3 Ab.

FIG. 19 is a scheme showing a physical association between GNAP andMCM3. Anti-GST, anti-GNAP (42-23) and anti-MCM3 immunoprecipitates fromWEHI-231 cell lysates were subjected to in vitro kinase assay. Normalrabbit serum (NRS) was used as a control for anti-MCM3 Ab. The sampleswere separated by 7% SDS-PAGE. The bands corresponding to GNAP and MCM3were indicated by arrows in the left panel. On the right panel, V8cleavage mapping of 210-kDa bands showed an identical cleavage pattern.As a control an irrelevant V8-digested protein was separated inparallel.

FIG. 20 is a scheme showing a result where double staining withanti-MCM3 Ab and anti-CR1 mAb, or PNA, was performed. The expression ofMCM3 was upregulated in GC area.

FIGS. 21A-B are schemes where a deduced amino acid sequence of humanGANP protein is represented in one character notation SEQ ID No: 3.

FIG. 22 is a photograph showing a result where human ganp and Map 80were mapped by FISH method using human chromosome.

PREFERRED EMBODIMENT FOR CARRYING OUT THE INVENTION

The typical examples of GANP protein of the present invention areprotein represented by the amino acid sequences of SEQ ID No. 1 and No.3 of the sequence listing, and are characterized in that they have amolecular weight of 210 kDa and have a kinase activity. GANP mutantproteins provided by the present invention are represented by the aminoacid sequences wherein approximately 1 to several, preferably 1 to 20,more preferably 1 to 10, most preferably 1 to 5 amino acid residues aresubstituted, inserted, and/or deleted in the amino acid sequences of SEQID No. 1 or No. 3, and have a kinase activity which is substantiallysimilar with GANP protein represented by the amino acid sequences of SEQID No. 1 or No. 2. These GANP mutant proteins are within the scope ofthe present invention. The protein represented by the amino acidsequences of SEQ ID No. 1 or No. 3 of the sequence listing and homologuethereof are those whose expression is selectively increased incentrocytes of germinal center of mammal from which the protein isderived.

Usually, the active domain of GANP protein or GANP mutant protein can bereadily identified by preparing a polypeptide wherein amino acidresidue(s) are deleted from N-terminal and/or C-terminal of the fulllength amino acid sequence, and measuring the kinase activity of thepolypeptide. The polypeptides provided by the present invention arethose comprised of an active domain of GANP protein and GANP mutantprotein and those comprising, as a partial sequence, a polypeptidecomprised of said active domain, and have a kinase activity which issubstantially similar with GANP protein. Moreover, another polypeptidesprovided by the present invention are those comprising, as a partialsequence, a full length amino acid sequence of GANP protein or GANPmutant protein, and have a kinase activity which is substantiallysimilar with GANP protein.

The polynucleotide provided by the present invention includes DNA andRNA as well as all of the nucleotides obtained by chemically modifyingDNA or RNA. The term “polynucleotide” used herein should be most broadlyinterpreted to include non-naturally occurring form. The typicalexamples of the polynucleotide provided by the present invention are DNAor RNA which encodes the aforementioned GANP protein or GANP mutantprotein. Another example of the polynucleotide of the present inventionis antisense polynucleotide.

It is well known for a skilled person in the art that, using degeneracyof genetic code, at least partial bases of a polynucleotide can bereplaced with another type of bases without changing the amino acidsequence of the polypeptide which is produced from the polynucleotide.Therefore, the polynucleotide of the present invention includes allpolynucleotides which encode GANP protein or GANP mutant protein. Asexamples of the preferred gene of the present invention, a gene encodingGANP protein from mouse is shown in SEQ ID No.2 of the sequence listing,and a gene encoding GANP protein from human is shown in SEQ ID No.4. Theamino acid sequence of GANP mutant protein can be determined from thebase sequence of a gene encoding said mutant. For example, sequencingcan be carried out by using commercially available programs (forexample, MacVector (registered trademark, Eastman Chemical), or Genetix(Software Kaihatsu)).

The scope of the present invention covers antisense polynucleotidescomposed of a base sequence of antisense chain of polynucleotideencoding GANP protein, and derivatives thereof. The antisensepolynucleotides is provided as an embodiment of the polynucleotidementioned above, and the term “antisense polynucleotide” may be hereinused to clearly mean that it is a polynucleotide comprised of basesequence of antisense chain. The antisense polynucleotide can hybridizeto polynucleotide encoding GANP protein, and if the polynucleotide towhich it hybridize is a polynucleotide of coding region, thebiosynthesis of the polypeptide encoded by the polynucleotide can beinhibited.

Antisense polynucleotide for inhibiting the biosynthesis of polypeptidepreferably contains 12 or more bases. On the other hand, anunnecessarily long sequence is not preferred in order to incorporatefull length antisense polynucleotide into cells. When an antisensepolynucleotide is incorporated into cells to inhibit the biosynthesis ofGANP protein, it is preferred to use an antisense polynucleotide of 12to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases.

The antisense polynucleotide of the present invention or derivativesthereof include all of the form where several nucleotides composed ofbase, phosphoric acid and sugar are bound whether or not they arepresent in nature. Typical examples include a naturally occurringantisense DNA and antisense RNA. Non-naturally occurring polynucleotidesinclude, for example, polynucleotides of methylphosphonate type andphosphorothioate type. As to the antisense polynucleotide of the presentinvention, various antisense polynucleotide derivatives which areexcellent in binding ability to target DNA or mRNA, tissueselectability, cell permeation property, nuclease resistance,intercellular stability and the like, can be obtained by using anantisense technology available to a skilled person in the art.

Generally, in view of easiness of hybridization, it is preferred todesign an antisense polynucleotide or derivatives thereof having a basesequence complementary with base sequence which forms a loop of RNA.Therefore, as to the antisense polynucleotide of the present inventionand derivatives thereof, those which hybridize to loop region of RNA arepreferred examples. Moreover, an antisense polynucleotide having asequence complementary with a translation initiation codon andneighborhood thereof, ribosome binding site, capping site, or splicingsite can generally be expected to exhibit high expression inhibitioneffect. Therefore, the antisense polynucleotide of the present inventionor derivatives thereof having a sequence complementary with atranslation initiation codon and neighborhood thereof ribosome bindingsite, capping site, and/or splicing site of the gene encoding GANPprotein are preferred example in view of expression inhibition effect.

Among the currently generally known polynucleotide derivatives, thederivatives where at least one of nuclease resistance, tissueselectability, cell permeation property and binding ability is enhanced,preferably include derivatives having a phosphorothioate bond as askeleton structure. The polynucleotide of the present invention andderivatives thereof include derivatives having these function orstructure.

Among the antisense polynucleotide of the present inventions naturallyoccurring type of antisense polynucleotide may be synthesized by using achemical synthesizer or may be prepared by a PCR method using a DNAencoding GANP protein as a templeta. Polynucleotide derivatives such asmethylphosphonate type or phosphorotioate type may usually be preparedby chemical synthesis. In this case, the procedure can be carried outaccording to an instruction attached with the chemical synthesizer, andthe synthesized product thus obtained can be purified by HPLC methodusing reverse phase chromatography and the like.

Polynucleotide which is a polynucleotide encoding GANP protein of thepresent invention, antisense polynucleotide thereof, or a portionthereof (for example, polunucleotide composed of continuous 12 or morebases) can be used as a probe for screening a DNA encoding GANP proteinfrom mammalian cDNA library. For such a purpose, a polynucleotidecomposed of a sequence of continuous 15 or more bases is particularlypreferred. The polynucleotide used as a probe may be a derivative.Usually, it is recognized that a sequence having the aforementionednumber or more of base is a specific sequence.

ADNA of continuous 12 or more bases in the base sequence of SEQ ID No. 2or No. 4 of the sequence listing, or a polynucleotide which hybridizesto said DNA (antisense polynucleotide) can be used as a probe forscreening a DNA encoding GANP protein from cDNA library or the like.

Also, a tissue which expresses mRNA from GANP gene can be detected byperforming a Northern Blot hybridization on mRNA derived from varioustissues by using a polynucleotide encoding GANP protein of the presentinvention, antisense polynucleotide thereof or a polynucleotide of aportion thereof as a probe. Furthermore, a polynucleotide of 12 or morebases can be used as a primer for polymerase chain reaction (PCR), and apolynucleotide encoding GANP protein can be obtained by PCR. Also, theprimer can be appropriately selected to clone any portion of GANPprotein.

As to the cDNA library used in the screening using the aforementionedprobe, one prepared from mRNA can be preferably used. A group of cDNAselected by random sampling from these cDNA library may be used as asample for screening. A commercially available cDNA library can be used.

The cDNA which hybridizes to the above-obtained GANP gene is insertedinto a suitable vector (for example, pGEX-4T-1 vector), and isintroduced into a host (for example, E. coli) to prepare a transformant.The type of the vector and the type of the host are not particularlylimited, and any suitable expression vector may be selected and useddepending on the type of the host. As the host, bacterium such as E.coli, yeasts, or animal cells can be used. A method for obtaining atransformant by introducing a recombinant vector into a suitable hostsuch as E. coli is not particularly limited, and any method available toa skilled person in the art may be applied.

The transformant into which the GANP gene of the present invention wasintroduced can be cultured to amplify a gene DNA or produce a protein,thereby prodicing GANP protein. The preparation and culturing of atransformant are described in various literatures and reports, and manymethods have been developed and have been conventionally used in theart. Therefore, a skilled person in the art can easily prepare GANPprotein on the basis of the base sequence described herein. The methodsfor introducing a gene into cells include calcium chloride method,lipofection method, protoplast method, and electroporation method.

Separation and purification of a protein of interest from the culturecan be carried out by using any means available to a skilled person inthe art in combination appropriately. For example, GANP protein of thepresent invention can be efficiently recovered and purified byperforming procedures such as concentration, solubilization, dialysis,various chromatography and the like. More specifically, selection may besuitably made among immunoprecipitation, salting out, ultrafilteration,isoelectric point precipitation, gel filteration, electrophoresis,various chromatography such as ion exchange chromatography, hydrophobicchromatography and antibody chromatography, chromatofocusing, adsorptionchromatography, and reverse phase chromatography. By using a geneencoding GANP mutant protein, GANP mutant protein can be similarlyprepared.

Also, GANP protein or GANP mutant protein can be prepared as a fusedprotein with another polypeptide. Such a fused polypeptide is within thescope of the present invention. The type of the polypeptide to be fusedis not particularly limited, and includes, for example, a signal peptidewhich promotes an extracellular secretion. The preparation of such afused protein may be carried out by using transformant. When a fusedprotein is used to prepare GANP protein or GANP mutant protein, a fusedprotein is treated with a chemical substance such as bromecyan or anenzyme such as protease, and the substance of interest which was cut outmay be separated and purified.

Antibodies which recognize GANP protein or GANP mutant protein can beprepared by using GANP protein or GANP mutant protein of the presentinvention or partial polypeptide thereof. The antibody of the presentinvention can be prepared by any means of a conventional method in theart by immunizing a mammal with GANP protein or GANP mutation protein.It can be confirmed by Western blotting, ELISA, immunostaining (forexample, measurement with FACS) or the like that the antibody recognizesGANP protein or GANP mutation protein of the present invention. Asimmunogens, there may used GANP protein or GANP mutant protein as wellas a portion thereof bound to another carrier protein such as calf serumalbumin. A portion of GANP protein or GANP mutation protein preferablycontains 8 or more amino acid residues, and such a polypeptide may besynthesized by using, for example, a peptide synthesizer.

A monoclonal antibody which is produced from hybridoma prepared by usinglymphocytes of immunized animals may be used as an antibody of thepresent invention. The process for the preparation of a monoclonalantibody is well known in the art and is conventionally used(“Antibodies, A Laboratory Manual”(Cold Spring Harbor Laboratory Press,1988), Chapter 6). Moreover, a fragment of antibody having aantigen-antibody reaction activity and a chimera antibody may be used asan antibody of the present invention. GANP protein or GANP mutantprotein of the present invention can be detected by a method using anantibody or a method using an antibody and an enzyme.

The present invention is illustrated in detail by the examples below,but the scope of the present invention is not limited to the examplesbelow.

EXAMPLE Example 1 Cloning of Mouse GANP Gene and Analysis of Expression

<Materials and Methods>

(1) Animals and Immunization

BALB/c mice and Lewis rats were purchased from Seac Yoshitomi Ltd.(Fukuoka). NZB, NZW, (NZB×NZW)F₁ mice (7 week old, female), MRL/lpr mice(8 week old, female), and BXSB mice (7 week old, male) were obtainedfrom Japan SLC Co. (Shizuoka). Aged NZB mice (10 month old, female) werekindly gifted from Dr. Sachiko Hirose (Department of Pathology, JuntendoUniversity School of Medicine). NOD mice (7 week old, male) weregenerously provided from Dr. Junichi Miyazaki (Department of Nutritionand Physiological Chemistry, Osaka University Medical School). Allanimals were maintained in Center for Animal Resources and Developmentin Kumamoto University. BALB/c mice were immunized multiply with sheepred blood cells (Nippon Bio-Test Laboratories, Inc., Tokyo). Theimmunization was performed intravenously with 5-day interval andsections of the thymus, spleen, lymph node (LN), and Peyer's patches(PP) were prepared for the immunohistochemical analysis.

(2) Cells and Cell Culture

Splenic B cells from BALB/c mice were enriched as described previously(Nomura et al., Immunol. Lett. 45:195-203, 1995). These cells werecultured in RPMI-1640 medium (Gibco-BRL, Gaithersburg, Germany)containing 10% heat-inactivated FCS (Dainippon Pharmaceutical Co.,Osaka, Japan), 5 mM L-glutamine (Biowhitteker, Walkersville, Meryland,USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-ME at 37°C. in an incubator with 5% carbon dioxide.

(3) Establishment of the 29-15 Monoclonal Antibody (Hereinafter Referredto as “25-15 mAb”)

The mAbs against a murine B cell line WEHI-231, which was establishedfrom a (BALB/c×NZB)F₁ mouse with mineral oil were prepared by the methoddescribed previously (Kuwahara et al., J. Immunol. 152:2742-2752, 1994).Briefly, the cell lysate of WEHI-231 with the surface phenotypeslgM⁺slgD⁺B220⁺ was prepared with the hypotonic buffer in the absence ofdetergent and dialyzed against a phosphate buffered saline (PBS) inaccordance with the method of Sakaguchi et al (Sakaguchi et al., EMBO(Eur. Mol. Biol. Organ.) J. 5:2139-2147, 1986). The cell lysate wasimmunized into the foot pads of Lewis rats in the complete Freund'sadjuvant (CFA) (Difco Laboratories, Detroit, Mich., USA) and boostedtwice in the incomplete Freund's adjuvant (IFA) (Difco Laboratories) atday 4 and day 8. After 9 days, the lymph node of popliteal and inguinalregions were excised and the lymphoid cell suspension was prepared.Establishment of hybridomas, selection in the HAT media (Gibco-BRL), andrecloning of hybridoma clones were performed as described previously(Kuwahara et al., J. Immunol. 152:2742-2752, 1994). The 29-15 mAb wasselected to stain lymphoid cells in the immunohistochemical analysis.

(4) Antibodies and Reagents

F(ab′)₂ fragment of the affinity-purified goat anti-mouse μantibody (ICNPharmaceutical, Inc., Costa Mesa, California, USA), biotin-conjugatedpeanut agglutinin (PNA) (Vector Laboratories, Inc., Burlingame, Calif.,USA), biotin-conjugated anti-CD35 mAb (PharMingen, San Diego, Calif.),alkalinephosphatase (ALP) conjugated goat anti-rat IgAb (#59301, ICN),HRP conjugated goat anti-rat IgAb (ICN), HRP conjugated streptavidin(Kirkegaard & Perry Laboratories, Inc., Gaitherburg, Md), ALP conjugatedgoat anti-mouse IgAb (Sigma Chemicals Co., St. Louis, Mo.), FITCconjugated mouse anti-rat κmAb (ICN), PE conjugated anti-B220 mAb(PhaMingen), and ALP conjugated goat anti-rabbit IgAb (ZymedLaboratories Inc., South San Francisco, Calif.) were purchased and used.Biotin-conjugated mAbs such as anti-B220 (RA3-6B2), anti-μ(AM/3), andanti-δ(CS/15) were prepared in our laboratory. Anti-CD40 mAb (LB429) wasestablished in our laboratory (Nomura et al., Immunol. Lett. 45:195-203,1995). Hybridomas of AM/3 and CS/15 were kindly provided by Dr. KensukeMiyake (Department of Immunology, Saga Medical School).Biotin-conjugated anti-Syndecan-1 was purchased from PharMingen (SanDiego, Calif., USA). Anti-BrdU mAb was obtained from NovocastraLaboratories, Ltd. (Newcastle, United Kingdom). Rabit anti-mouse MCM3/P1Ab is described in the literature(Kimura, H et al, 1994, EMBO J. 13,4311-4320).

(5) Immunohistochemistry

Immunohistochemical staining was performed as described previously(Ezaki et al., Arch Histol. Cytol. 58:104-115, 1995; Yamanouchi et al.,Eur. J. Immunol. 28:696-707, 1998). In brief the target organs excisedfrom BALB/c, NZB, (NZB×NZW)F₁, NOD, BXSB, and MRL/pr mice were placed inOCT compound (Miles Inc., Elkhart, Indiana, USA). The 6-μm cryosectionsplaced on the gelatin-coated slides were air-dried fully. The slideswere then fixed in acetone for 10 minutes, followed by rehydration inPBS for 15 minutes. The slides were incubated with the 29-15 mAb for 60minutes and were washed with PBS several times. After incubation withalkaline phosphatase-conjugated goat anti-rat lg antibody (ALP-anti-ratlg, catalogue #59301, ICN Pharmaceutical Inc.), the slides were washedfour times with PBS. The slides were developed using Vector Blue (VectorLaboratories).

For secondary staining, the slides were incubated with biotin-labeledmAbs in combination with horseradish peroxidase (HRP)-conjugatedstreptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md,USA). After development with p-dimehylaminoazobenzene (DAB, Dojindo,Kumamoto), the sections were fixed lightly with 1% glutaraldehydesolution in PBS. To detect the cells of active proliferation in vivo,BrdU (Sigma Chemicals Co., St. Louis, Mo., USA) was injectedintravenously 1 hour before obtaining the organs. Cells undergoing DNAsynthesis were detected by staining with anti-BrdU mAb in combinationwith ALP-conjugated goat anti-mouse lg Ab (Sigma Chemicals Co.) followedby development with Vector Red (Matsuno et al., Cell Tissue Res.257:459-470, 1989). Periodic acid Schiff (PAS) staining was performed asdescribed previously (Jiang et al., J. Immunol. 158:992-997, 1997). Allsections were mounted by Aquatex (E. Merck, Darmstadt, Germany).

(6) Molecular Cloning of the cDNA Using λgt11 Vector

The cDNA libraries constructed with mRNAs from the mouse spleen, mousebone marrow, WEHI-231 cells and A20 cells were screened with thesupernatant of the 29-15 mAb after transferring the fusion protein ontonitrocellulose filters (Schleicher and Schuell, Darmstadt, Germany) thatwere presoaked with 20 mM IPTG (Inui et al., J. Immunol. 154:2714-2723,1995). The phage plates were incubated for 4 hours at 42° C. and thenthe plates were covered with the filters and further incubated for 4hours at 37° C. The filters were washed three times with the washingbuffer (PBS containing 0.1% Tween 20), blocked for 1 hour in theblocking buffer (5% nonfat dry milk in PBS containing 0.1% Tween 20),and then incubated with the 29-15 mAb. Positive signals were detected byautoradiography using ¹²⁵l-labeled sheep anti-rat lg Ab (Amersham,Buckinghamshire, United Kingdom). The initial cDNA clone contained a280-bp fragment that is capable of coding a polypeptide as a fusionprotein. With the original 280-bp fragment, the longer cDNA clones wereisolated from another WEHI-231 cDNA library. The 4.9-kb fragment of thesecond cDNA clone encodes a longest open reading frame of 4.5 kb. Tofurther determine the 5′ sequence, the 5′-RACE method was employed. Therace kit of Gibco-BRL was used.

(7) In situ RNA Hybridization on Tissue Sections

In situ RNA hybridization was carried out as described previously (Kondoet al., Blood 80:2044-2051, 1992). Paraffin-embedded sections weremounted on silanized slides. After the slides were deparaffinized,hybridization with ganp 280-bp riboprobe labeled by digoxigenin wasperformed for 16 hours at 50° C. The slides were washed with TNE buffer(10 mM Tris-HCl [pH 7.6], 500 mM NaCl, 1 mM EDTA) at 37° C. severaltimes, followed by washing with 2X and/or 0.2X SSC solution at 50° C.While using anti-digoxigenin antibody, the development was performed inthe presence of ALP substrate.

(8) Preparation of GST-cDNA Fusion Protein and Another Anti-GANP mAb

The ganp cDNA fragment encoding a part of GANP (amino acids of 679th to1028th of the amino acid sequence of SEQ ID No. 1 of the sequencelisting) was introduced into a pGEX-4T-1 vector (Pharmacia Biotech,Piscataway, N.J., USA). The recombinant plasmid was verified by DNAsequencing of the entire insert and the junction. The GST-GANP fusionprotein was prepared by glutathione-Sepharose (Pharmacia) columnchromatography as described elsewhere (Inui et al., J. Immunol.154:2714-2723, 1995). Anti-GANP mAb, designated 42-23, was establishedby immunizing the fusion protein in rats as described above.

(9) Western Blot Analysis

Protein gel electrophoresis, Western blot transfer, and theimmunodetection of proteins were performed as described previously(Kuwahara et al., Int. Immunol. 8:1273-1285, 1996). Fifty million cellswere lysed with 1 ml of the TNE lysis buffer (10 mM Tris-HCl [pH 7.8],150 mM NaCl, 1 mM EDTA, 1% NP40, 0.02% NaN₃) and the immune complex wasanalyzed on SDS-PAGE (7%). After the proteins were transferred onto anitrocellulose filter, the filter was blocked with PBS-Tween 20containing 5% nonfat dry milk and incubated with anti-GANP mAb for 60minutes. After washing with PBS-Tween 20 several times, the filter wasincubated with HRP-conjugated goat anti-rat lg (ICN Pharmaceutical Inc.)for 30 minutes. The development was performed using an ECL detection kit(Amersham).

(10) Subcellular Fractionation

Separation of intact nuclei was carried out as described previously(Schriber et al., Nucleic Acids Res. 17:6419, 1989). WEHI-231 cells werewashed with TBS and the pellets were resuspended in buffer A (10 mMHEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mMPMSF) and incubated for 15 minutes on ice, followed by the addition ofNP-40 to a final 1%. After the centrifugation, the supernatants wererecovered as a cytoplasmic fraction. The pellets were resuspended withthe same buffer and homogenized to obtain the intact nuclei by staining.The sample was centrifuged and the pellet was resuspended with coldbuffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMDTT, 1 mM PMSF) and centrifuged. The supernatants were frozen at −80° C.as a nuclear fraction.

(11) In Vitro Kinase Reaction and Phosphoamino Acid Analysis

Kinase reaction was carried out in vitro with the immunoprecipitate asdescribed previously (Kuwahara et al., J. Immunol. 152:2742-2752, 1994).Splenic B cells were purified by the method described (Nomura et al.,Immunol. Lett. 45:195-203, 1995). The B cells were stimulated in vitrofor 48 hours with F(ab′)₂ fraction of goat anti-IgM Ab and anti-CD40 mAb(LB429) as described previously (Nomura et al., Immunol. Lett.45:195-203, 1995). After harvesting and washing, cells were lysed withTNE lysis buffer and immunoprecipitated with the anti-GANP mAb (42-23).The immunoprecipitates were incubated with [γ-³²P]-ATP (Amersham) andthe radiolabeled proteins were analyzed on SDS-PAGE (7%) and withautoradiography. The band corresponding to GANP was excised from driedgel. After SDS was removed from the gel the homogenized gel was digestedby TPCK-trypsin (Sigma Chemicals Co.) at 37° C. overnight. The sampleswere subjected to hydrolysis with 6N HCl and electrophoresed onto TLC(E. Merck).

V8 cleavage mapping of the indicated proteins was carried out asdescribed previously (Kuwahara, K, et a 1994, J. Immunol.152:2742-2752).

(12) Cytoplasmic Staining

The cells were fixed with 2.5% paraformaldehyde solution in PBS followedby permeabilization with 70% ethanol for 1 hour on ice. The cells wereincubated with the 29-15 mAb in combination with FITC-conjugated mouseanti-rat κ mAb. Antibody-binding was analyzed on FACScan flow cytometer(Becton-Dickinson, Mountain View, Calif., USA).

(13) Immunoprecipitation and Western Blot Analysis

Proteins obtained in the aforementioned (10) subcellular fractionationwere immunoprecipitated with the anti-GABP mAb in combination withprotein G-Sepharose, and analyzed by SDS-PAGE. The Western blot filterwas incubated with the anti-GANP mAB, followed by HRP-anti-rat lg. Thedevelopment was performed using an ECL detection kit (Amersham).

(14) Reverse Transcriptase-PCR (RT-PCR)

Total RNA (1 μg each), purified from cultured B cells using TRISOL(Gibco BRL, Rockville, Md.) was used as a template for the cDNAsynthesis (100 μl volume) with Superscript (Gibco BRL). PCRamplification was carried out using 2 μl of each cDNA solution withTaq-Gold (Perkin-Elmer, Foster, Calif.) and the primers for ganp or HPRT(control) (Han, S., et al, 1996, Science. 274-2092-2097). The ganptranscripts were amplified by 5′-CCGTGGGATGACATCATCAC-3′ (the forwardprimer) (SEQ ID No. 5 of the sequence listing) and5′-CATGTCCACCATCTCCAGCA-3′ (the reverse primer) (SEQ ID No. 6 of thesequence listing).

<Results>

(1) Expression of the GANP Antigen in Lymphoid Organs

An mAb that recognizes a differentiation antigen expressed in peripheralB cells was prepared by immunizing rats with the lysate of WEHI-231cells. Immunohistochemical analysis with the 29-15 mAb on normallymphoid organs of BALB/c mice did not detect expression in the bonemarrow, but showed the slight expression in lymphoid organs such as thethymus, spleen, and lymph node. A small number of cells in the red pulpof the spleen and the deep cortex of the lymph node strongly express the29-15 Ag. Interestingly, the expression was very high in the centralarea of follicles of the PP (FIG. 1). The cells were positive withanti-B220 mAb, but not with anti-lgD mAb. Normal mice show thedevelopment of secondary lymphoid follicles with clear GC in PP becauseof the continuous stimulation of various antigenic substances introducedthrough the intestinal lumen.

Repeated immunization with sheep red blood cell (SRBC) induces theformation of lymphoid follicles in the spleen within 12 days. Antigenimmunization induces an appearance of 29-15⁺ cells in the GC area of thespleen and lymph node as well as in the GC of the PP (FIG. 2). The 29-15antigen appeared upregulated in cells of the GC. The phenotype of 29-15⁺cells in the architecture of secondary lymphoid follicles was furtheranalyzed. Nearly half of PNA⁺ GC-B cells are positive with the 29-15mAb, but they are negative with anti-BrdU mAb (FIG. 3). Interestingly,the expression of 29-15 Ag is upregulated in the centrocyte area at thedistal region of the entrance from the central artery. This phenotype isconsistent with the criteria of GC-B cells and supports the name “GANP”for the 29-15 Ag as described above.

(2) Appearance of GANP^(dense+) B Cells in the Red Pulp Area ofAutoimmune-Prone NZB Mice

Normal mice express few GANP⁺ B cells in the follicular area of thespleen without in vivo stimulation but show a few GANP^(dense+) cellswhich remarkably express GANP protein in the red pulp area of BALB/c(FIG. 2) and C57BL6. These cells are large and obviously different fromconventional B cells. In young (8 week old) NZB mice, however, theseGANP^(dense+) cells increased spontaneously in the red pulp area of thespleen without immunization (FIG. 4). Another autoimmune-prone mouse,NZW, does not express GANP^(dense+) cells in the red pulp at ages of 5to 12 weeks. A severe-disease combination of (NZB×NZW)F₁ shows anintermediate expression of GANP^(dense+) cells in the red pulp.

Whether the GANP^(dense+) cells also appear spontaneously in the spleenof other autoimmune-prone mice was examined. The GANP^(dense+) cellsappear in the spleen of BXSB and MRL/lpr, but not markedly in NZW andNOD mice at a similar age in the specific pathogen free condition (SPF).The GANP^(dense+)cells become apparent during aging and appear in theperipheral lymph node of the aged-NZB mice (10 month old) that havepassed the onset of the disease. The appearance of the GANP^(dense+)cells in the lymph node seems to be mostly in the later stage. Ofparticular interest, MRL/lpr shows the appearance of GANP^(dense+) cellsin the lymph node at the young stage (8 week old)(FIG. 5). These resultssuggested that a genetic factor in autoimmune-prone NZB, BXSB, andMRL/lpr mice might control the appearance of GANP^(dense+) cells in thered pulp area and the recruitment into the lymph node.

Two-color analysis showed the phenotype of GANP^(dense+)cells in the redpulp area as PNA⁻B220⁻ cells (FIG. 6) and IgD-CD38⁻ cells. These cellsare positive when stained with anti-Syndecan-1 mAb, which stains plasmacells selectively. The GANP^(dense+) cells express lgM in cytoplasm(FIG. 7). Because these cells could be Mott cells (Jiang, Y., S. Hirose,Y. Hamano, S. Kodera, H. Tsurui, M. Abe, K. Terashima, S. Ishikawa andT. Shirai. 1997. J. Immunol. 158:992-997.), the section was stained withPAS staining. The GANP^(dense+) cells show PAS⁻, as with theB220⁻Syndecan-1⁺PNA⁻BrdU⁻GANP^(dense+) (FIG. 8) and CD40⁻CD38⁻. Theseplasma-like cells appear preferentially in the spleen of NZB mice, butare different from Mott cells currently reported.

(3) Identification of a cDNA Clone Encoding the GANP Antigen

Using the 29-15 mAb, we isolated a candidate cDNA clone (with the insertDNA of 280 bp) from the WEHI-231 cDNA library and further isolated alonger cDNA clone, named ganp. The full-length nucleotide sequence (6429bp) determined from overlapping clones shows a putative polypeptidecomposed of 1971 amino acids with a predicted molecular size of 210-kD(FIG. 9). The amino acid sequence of GANP protein is shown in SEQ ID No.1 of the sequence listing and the base sequence of ganp cDNA is shown inSEQ ID No. 2 of the sequence listing.

The GANP amino acid sequence shows a regional homology to SAC3 which isconsidered to be a nuclear transcription regulation factor characterizedin temperature-mutant Saccharomyces cerevisiae and human Map 80 protein(Takei, Y et al, 1998, J. Biol. Chem. 273:22177-22180) (FIG. 10 and FIG.15; Bauer, A. and R. Koelling, 1996. Yeast 12:965-975). The GANP proteinshows mild homologies within short stretches of the insulin promoterfactor (amino acids 996 to 1063) and various transcription factors,including NF-IL-6 (amino acids 388 to 450).

The GANP gene shows a consensus base sequence for the super coil motifs,but does not show zinc-finger, leucine-zipper, and homeo-domain motifs.A serine/threonine-rich region was seen in N-terminal 100 amino acids,which has slight homology to nucleoporin, which is known as the nuclearpore complex. GANP has two possible nuclear localization sequences(⁴⁹⁷HKKK and ¹³⁴⁴PMKQKRR), which would potentially support theexpression of the GANP in the nucleus as suggested by the PSORT program.Moreover, GANP has 2 coiled-coil motifs, but does not have zinc-finger,leucine-zipper, and homeo-domain motifs. Further, there were 4 LXXLLmotifs which were recognized in nuclear transcription coactivatormolecules including CBP/p300 and p/CIP (Torchia, J. et al., 1997. Nature(Lond.) 387:677-684; Heery et al., 1997, Nature (Lond.) 387:733-736),but any association molecule through these motif. has not beenidentified.

(5) Expression of the ganp Transcripts

Northern blot analysis detected the 7-kb mRNA as a very weak signal incomparison to the control β-actin signal, but its expression was ratherubiquitous in all cell lines, organs, and tissues tested. In order toexamine whether the ganp mRNA is upregulated in the same areas asdetected on sections with the 29-15 mAb, in situ RNA hybridizationanalysis was carried out. The ganp mRNA is expressed abundantly in thecentral area of the GC of the SRBC-immunized spleen, but not in thenonimmunized spleen (FIG. 11), thymus, and lymph node. The ganp mRNA wasupregulated in GC-B cells of immunized mice. This expression pattern isquite similar to the results with the 29-15 mAb on the same sectionbased on staining with hematoxylin. The GC area of the PP also showedupregulation of the ganp mRNA in nonimmunized BALB/c mice, and theexpression of ganp mRNA is high in plasma-like cells of the red pulparea of the spleen of nonimmunized NZB mice (FIG. 11). These resultssuggests that the ganp gene encodes a molecule recognized by the 29-15mAb.

(6) Expression of the GANP in B Cells

The anti-GANP mAb (42-23) detected a single protein band at 210-kD fromboth nuclear and cytoplasmic compartments of WEHI-231 cells (FIG. 12).In order to find evidence of the functional involvement of the GANP inthe activation and differentiation of B lineage cells, B cells fromnonimmunized BALB/c mice were stimulated in vitro with anti-lgM andanti-CD40 in combination, and as a result, an expression of the GANPprotein detected with the anti-GANP mAb was increased (FIG. 13). An invitro kinase reaction with the GANP immunoprecipitates showed anincreased kinase activity assembled with the GANP protein in spleen Bcells stimulated in vitro. Thus, the GANP protein is induciblyphosphorylated at the serine/threonine residues (FIG. 14). These resultssuggest that the GANP might play a role to the activation of B cells inperipheral immune responses.

Stimulation with anti-μ Ab and anti-CD40 mAb showed maximal response,but either one of these regents showed only a marginal response (datanot shown). This upregulation was also detected by the increase of ganpmRNA in B cells stimulated by anti-μ and anti-CD40 co-ligation in vitro(FIG. 16). RT-PCR clearly demonstrated that the amount of ganp mRNAincreased at 24 hours and 48 hours after stimulation in comparison withthe control HPRT mRNA.

Since the 210-kDa GANP has many possible phhosphorylation sites, weexamined the induction of phosphorylation by an in vitro kinase reactionwith anti-GANP immunoprecipitates. As shown in FIG. 17, phosphorylationof the 210-kDa protein was found in the anti-GANP immunoprecipitatesfrom spleen B cells stimulated by anti-μ and anti-CD40 co-ligation. Thisresult indicates that a kinase activity is maintained even if GANP isprecipitated.

(7) Association of GANP with MCM3 Protein

We found a Map80-homologous region (76.3% identity at amino acid level)in the carboxyl-terminal part of GANP. Map80 is an 80-kDa nuclearprotein that is involved in the translocation of MCM3 (a proteinessential for DNA replication) between the cytoplasm and the nuclei(Takei, Y. et al, 1998, J. Biol. Chem. 273:22177-22180; Kimura, H. etal, 1994, EMBO J. 13:4311-4320; Chong, J. P. et al. 1996, Trends.Biochem Sci 21:102-106; and Romanowski, P et al., 1996, Curr. Biol.6:1416-1425). Therefore, we examined the interaction between GANP andMCM3 in WEHI-231. We detected that anti-GANP immunoprecipitates includeMCM3. Because the phosphorylation states of MCM proteins seem crucial inregulation of cell cycle progression (Kimura, H. et al, 1994, EMBO J.13:4311-4320; Chong, J. P. et al, 1996, Trends Biochem Sci. 21:102-106;and Romanowski, Pet al, 1996, Curr. Biol. 6:1416-1425), in vitro kinaseassays with anti-MCM3 immunoprecipitates was performed.Immunoprecipitation of MCM3 co-precipitated a phosphorylated proteinmigrated at 210-kDa, which is the identical size of GANP (FIG. 19, leftpanel). These 210-kDa bands from anti-GANP and anti-MCM3immunoprecipitates showed an identical pattern in the V8 cleavagemapping (FIG. 19, right panel), indicating that GANP and MCM3 areassociated in a B cell line.

Next, we studied whether MCM3 is upregulated in GC-B cells byantigen-immunization of mice in vivo. The contiguous sections to thoseused above were stained with the anti-MCM3 Ab (FIG. 20). MCM3 is alsoupregulated in GCs. Double staining clearly demonstrates theco-localization of both MCM3 and PNA. A part of GC area is surroundedintensely with FDCs (lymph follicular cells). These results demonstratethat MCM3 is upregulated in GC-B cells including centroblasts and theGANP⁺ centrocytes that would be mostly surrounded by FDCs (FIG. 20).

(8) Discussion

As mentioned above, the present inventors found a novel protein, GANP,expressed in GC-B cells localized at the light zone of secondaryfollicles in the spleen. Although a trace amount of the ganp mRNA isdetectable in many kinds of cells under normal conditions, the GANPprotein appears upregulated in the specified GC area of immunized mice.A number of studies demonstrated various differentiation antigens in theGC as molecules recognized with mAbs or by specific cDNA cloning(Christoph et al., Int. Immunol. 6:1203-1211, 1994; Li et al., Proc.Natl. Acad. Sci. USA 93:10222-10227, 1996; Kuo et al., J. Exp. Med.186:1547-1556, 1997). Most molecules appear in GC-B cells of the wholearea, whereas 8-oxoguanine DNA glycosylase is expressed in the dark zone(Kuo et al., J. Exp. Med. 186:1547-1556, 1997).

Interestingly, the GANP antigen is selective in the centrocyte of thelight zone. Recent studies have shown that RAG protein which isnecessary for rearrangement of immunoglobulin gene is selectivelyexpressed in centrocytes at the light zone (Hikida et al., 1996. Science(Wash. DC) 274:2092-2094, 1996; Han et al., Science (Wash. DC)274:2094-2097, 1996). Since the GC area probably provides the site forsecondary lg gene rearrangement occurring during T cell-dependentantibody responses, as described by Papavasiliou et al. and Han et al.(Papavasiliou et al., Science (Wash. DC), 278:298-301, 1997; Han et al.,Science (Wash. DC), 278: 301-305, 1997), the GANP protein might be acomponent associated with the maturation of antigen-specific B cells atthe centrocyte stage.

We found that the carboxyl-terminal portion of GANP has a significantsimilarity to human Map80, which facilitates the nuclear transport ofMCM3 (Takei, Y et al., 1998, J. Biol. Chem. 273:22177-22180).Immunoprecipitation experiments demonstrated that GANP also binds toMCM3 in WEHI-231. MCM3 is a member of the MCM protein family essentialfor the initiation of DNA replication (Kimura, H. et al. 1994, EMBO J.13:4311-4320; Blow, J. J. 1993. J. Cell Biol. 122.993-1002; Tye, B. K.1994. Trends Cell Biol. 4: 160-166; Chong, J. P. et al, 1996, TrendsBiochem Sci. 21:102-106; Romanowski, P et al, 1996, Curr. Biol.6:1416-1425; and Thommes, P et al. 1992, Nucl. Acids Res. 20:1069-1074). The major fractions of nuclear MCM proteins bind tochromatin at the beginning of the S phase, but dissociate duringreplication and accumulate as free proteins in the nucleosol. Therelease of MCMs from chromatin is accompanied by the phosphorylation ofseveral MCM proteins and their reassociation after mitosis isconcomitant with their dephosphorylation. It was suggested that MCMproteins are no longer synthesized in growth arrested, differentiatingcells and disappear with kinetics related to their half-life (Musahl,C., et al, 1998, Exp. Cell. Res. 241, 260-264). The MCM3 protein hasrecently been shown to an early target in apoptotic proteolysis (Schwab,B. L. et al., 1998, Exp. Cell. Res. 238:415-421). Schwab, B. L. et alproposed that active destruction of MCM3 inactivates the MCM complex andserves to prevent untimely DNA replication events during the executionof the cell death program. Our results showed that GC-B cells expresshigh level of MCM3, some of which is associated with GANP. However, itappears curious that a protein, upregulated in differentiated cells thatarrest the cell cycle, binds to another protein essential forprogression of the S phase. One possible speculation is that a functionof GANP may be inactivation of MCM3 through its binding. Theimmunohistochemistry data are consistent with the following idea; GANPis upregulated in growth-arrested centrocytes while MCM3 is expressedboth in rapid-cycling centroblasts and still in centrocytes in GCs.Although the amount of MCM3 would decrease by ceasing the geneexpression and active destruction (Musahl, C., et al, 1998, Exp. Cell.Res. 241, 260-264; and Schwab, B. L et al., 1998, Exp. Cell Res.238:415-421), inactivation of MCM3, which is still expressed incentrocytes, through the interaction with GANP could be anothermechanism to prevent DNA replication. In addition, both GANP and MCM3become phosphorylated with the co-precipitated kinase (FIG. 19). Sincethe highly phosphorylated MCM3 is thought to be inactivated form(Kimura, H. et al, 1994, EMBO J. 13:4311-4320), the association withGANP may stimulate phosphoryltion of MCM3.

The GANP protein has a close similarity to the SAC3 (SAC, suppressor ofactin) of yeasts, Saccharomyces cerevisiae, which was isolated in agenetic screen for suppressors of a temperature-sensitive mutation(act1-1) in the actin gene (FIG. 10; Novick et al., Genetics,121:659-674, 1989). The SAC3 protein is expressed in the nuclei and isrequired for normal progression of mitosis and protection against theloss of chromosomes (Bauer et al., J. Cell. Sci. 109:1575-1583, 1996).Null mutants of SAC3 grow very slowly and arc larger than wild-typecells. SAC3 participates in a process that affects both the actincytoskeleton and mitosis, which suggest that SAC3 regulates the geneexpression of actin or actin-binding proteins.

A gene (named LEP-1) that augments the transcription of the leucinepermease activity in Saccharomyces was identical to SAC3 (Stella et al.,Yeast 11:460-460, 1995). Although the LEP-1 gene induces theupregulation of the yeast leucine permease involved in selective aminoacid transport, the amino acid transport in eukaryotic cells, especiallythe molecules involved in amino acid permeation is not known(Mastroberardino et al., Nature (Lond.) 395:288-291, 1998). Although theSAC3/LEP-1 sequence does not show motifs homologous to a number oftranscription factors, the biological functions determined previously(Bauer et al., J. Cell. Sci. 109:1575-1583, 1996) suggest its regulatoryactivity of various target genes in the nucleus. The mouse GANP does notshow typical consensus motifs for nuclear transcription factors, but hasa common ancestor with SAC3 gene of yeasts and has structural similarityof possible phosphorylation sites, two nuclear localization sequences,and two super coil structures that might interact with othertranscription molecules.

GANP is selectively upregulated in centrocytes of Ag-immunized spleen.It is also useful as the differentiation marker to define the centrocytesubset that is closely interacting with FDCs in GC area. Our studyshowed that the BCR signal and the CD40 co-stimulation together causethe upregulation of GANP and lead to the signal transduction mediatedthrough GANP/MCM3 complex.

The defective gene in the autosomal recessive genetic disease autoimmunepolyendocrinopathy (APECED) is localized by linkage analysis to humanchromosome 21 (21q22.3), which encodes an AIRE gene product with apossible transcription regulator (Nagamine et al., Nature Genet.17:393-398, 1997). The autoantibody recognizes the AIRE proteinexpressed in the adrenal gland and other gonad-producing tissues.Studies of APECED drew an idea that the involvement of molecules withnuclear coactivator activity might be associated with the autoimmunity.Both the AIRE and GANP proteins do not have typical domains fortranscription regulators, but they have LXXLL motifs as similarlyobserved in nuclear transcriptional coactivators.

A B cell-specific nuclear coactivator (Bob1/OCA-B/OBF1) was recentlycharacterized as a cell-type-specific regulator of Oct1 and Oct2 (Luo etal., Mol. Cell. Biol. 15:4115-4124, 1995). The OCA-B targeted mice showthe impairment of the GC formation in the spleen after immunization withT-dependent antigen, which suggests the functional involvement of B cellmaturation in the GC area (Kim et al., Nature (Lond.) 383:542-547, 1996;Qin et al., EMBO J. 17:5066-5075, 1998). The expression of the GANPprotein might be under the control of the OCA-B cell in centrocytes. Themolecular interaction of the nuclear coactivator molecules would be animportant issue for the understanding of the B cell maturation in theGC.

The New Zealand model of SLE has been the experiment subject of genomelinkage studies to map the chromosomal positions ofdisease-susceptibility genes. At least 12 non-MHC loci linked withnephritis and autoantibody production such as on chromosome 4(designated Nba1), on chromosome 7, and on chromosome 1 (designated asNba2; Vyse et al., J. Immunol. 158:5566-5574, 1997) have beenindependently mapped. The GANP antigen on large cells is highlyupregulated in the red pulp area of the nonimmunized NZB mice (FIGS.4-8). NZB mice contained similar large lgM-producing cells, named Mottcells, in the red pulp area. Mott cells appear selectively in NZB and(NZB×NZW)F₁ mice, but not in normal BALB/c or C57BL/6 mice.

The precursor cells of Mott cells are probably B-1 B cells (Tarlinton etal., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J. Immunol.158:992-997, 1997), which suggests a close association with theautoimmunity of B cells. Mott cells are apparent with the inclusion bodyof lgM in the cytoplasm and positive staining with PAS (Tarlinton etal., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J. Immunol.158:992-997, 1997). Because GANP^(dense+) cells seem to be Mott cells,PAS staining was performed. However, GANP^(dense+) cells in the red pulparea of NZB mice are PAS⁻. The GANP^(dense+) lgM-producing cells appearin the spleen of NZB mice, as do Mott cells, but these cells aredifferent. The new type of lgM-producing cells could be generated by thepossible activation of an abnormal B cell population related to one ofthe chromosomal loci linked to disease-susceptibility.

Lyn^(-/-) mice and CD40L^(-/-) mice reported from several laboratoriesshow similar autoimmunities and hyper-lgM syndrome(s), which have anincreased appearance of immunoblast cells with the inclusion body in thespleen (Hibbs et al., Cell 83:301-311, 1995; Nishizumi et al., Immunity3:549-560, 1995; Xu et al., Immunity 1:423-431, 1994). Theseobservations suggest that the signal transduction through BCR and CD40is regulating the generation of the abnormal antibody-producing plasmacells. Stimulation of splenic B cells with anti-IgM and anti-CD40antibodies induces the phosphorylation activity of the GANP protein.This observation suggests that the GANP protein may be involved indownstream of the B cell activation site in the GC area and the abnormalB cell activation in NZB mice might be associated with the increasedexpression of GANP protein.

Example 2 Cloning of Human GANP Gene

On the basis of information of the sequence of rat GANP gene, human GANPgene was cloned and sequenced. Specifically, λ gt 11-human heart cDNAlibrary (Clontech) was used, and gsp1-1: TTTGTCTGGAGGATGATCGC (SEQ IDNo.7 of the sequence listing), gsp1-2:AAAGAGAAAGGGGCCAGGCC (SEQ ID No.8of the sequence listing) and gsp1-3:CCAGCTTCTTGTCCAAAAGC (SEQ ID No.9 ofthe sequence listing) were used as primers, and 5′ RACE System for RapidAmplification of cDNA Ends, Version 2.0(Gibco BRL) was used to carry outthe cloning and sequencing by a conventional method.

The base sequence of the obtained clone was determined. The basesequence of the obtained human GANP gene is shown in SEQ ID No.4 of thesequence listing. The amino acid sequence encoded by this base sequenceis shown in SEQ ID No.3 of the sequence listing and FIG. 21. Human GANPgene shows high homology with mouse GANP gene, and Human GANP containsMap80 domain of 80 kDa at carboxyl terminal.

In in situ RNA hybridization, ganp transcript seems to be activated atGC region of tonsil. GANP⁺ cells express CD38⁺IgD⁺ phenotype of memory Bcell.

These results show that human GANP is expressed also in GC-B cells ofsecondary lympho tissues. Moreover, since human GANP of 1980 amino acidshas a stretch of Map80 homologous region which binds to MCM3 protein inB cells, it is suggested that GANP is involved in the regulation of cellcycle in GC-B cells.

Furthermore, in situ hybridization was carried out by FISH method withthe obtained human GANP gene and human chromosome specimen. The resultsare shown in FIG. 22. As is understood from FIG. 22, the genome fragmentcontaining human GANP gene and Map80 was mapped on 22.3 of the long armof chromosome 21.

INDUSTRIAL APPLICABILITY

The protein of the present invention is a novel protein having a kinaseactivity, and may be involved in a signal conversion of abnormal B celldifferentiation in an autoimmune state. Therefore, the protein,polypeptide, polynucleotide, antisense polynucleotide and antibody ofthe present invention are useful for revealing the mechanism ofautoimmune.

9 1 1971 PRT Mouse 1 Met His Pro Val Asn Pro Phe Gly Gly Ser Ser Pro SerAla Phe Ala 1 5 10 15 Val Ser Ser Ser Thr Thr Gly Thr Tyr Gln Thr LysSer Pro Phe Arg 20 25 30 Phe Gly Gln Pro Ser Leu Phe Gly Gln Asn Ser ThrPro Ser Lys Ser 35 40 45 Leu Ala Phe Ser Gln Val Pro Ser Phe Ala Thr ProSer Gly Gly Ser 50 55 60 His Ser Ser Ser Leu Pro Ala Phe Gly Leu Thr GlnThr Ser Ser Val 65 70 75 80 Gly Leu Phe Ser Ser Leu Glu Ser Thr Pro SerPhe Ala Ala Thr Ser 85 90 95 Ser Ser Ser Val Pro Gly Asn Thr Ala Phe SerPhe Lys Ser Thr Ser 100 105 110 Ser Val Gly Val Phe Pro Ser Gly Ala ThrPhe Gly Pro Glu Thr Gly 115 120 125 Glu Val Ala Gly Ser Gly Phe Arg LysThr Glu Phe Lys Phe Lys Pro 130 135 140 Leu Glu Asn Ala Val Phe Lys ProIle Pro Gly Pro Glu Ser Glu Pro 145 150 155 160 Glu Lys Thr Gln Ser GlnIle Ser Ser Gly Phe Phe Thr Phe Ser His 165 170 175 Pro Val Gly Ser GlySer Gly Gly Leu Thr Pro Phe Ser Phe Pro Gln 180 185 190 Val Thr Asn SerSer Val Thr Ser Ser Ser Phe Ile Phe Ser Lys Pro 195 200 205 Val Thr SerAsn Thr Pro Ala Phe Ala Ser Pro Leu Ser Asn Gln Asn 210 215 220 Val GluGlu Glu Lys Arg Val Ser Thr Ser Ala Phe Gly Ser Ser Asn 225 230 235 240Ser Ser Phe Ser Thr Phe Pro Thr Ala Ser Pro Gly Ser Leu Gly Glu 245 250255 Pro Phe Pro Ala Asn Lys Pro Ser Leu Arg Gln Gly Cys Glu Glu Ala 260265 270 Ile Ser Gln Val Glu Pro Leu Pro Thr Leu Met Lys Gly Leu Lys Arg275 280 285 Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg His Cys His Glu AlaAla 290 295 300 Glu Asp Pro Asp Pro Leu Ser Arg Gly Asp His Pro Pro AspLys Arg 305 310 315 320 Pro Val Arg Leu Asn Arg Pro Arg Gly Gly Thr LeuPhe Gly Arg Thr 325 330 335 Ile Gln Glu Val Phe Lys Ser Asn Lys Glu AlaGly Arg Leu Gly Ser 340 345 350 Lys Glu Ser Lys Glu Ser Gly Phe Ala GluPro Gly Glu Ser Asp His 355 360 365 Ala Ala Val Pro Gly Gly Ser Gln SerThr Met Val Pro Ser Arg Leu 370 375 380 Pro Ala Val Thr Lys Glu Glu GluGlu Ser Arg Asp Glu Lys Glu Asp 385 390 395 400 Ser Leu Arg Gly Lys SerVal Arg Gln Ser Lys Arg Arg Glu Glu Trp 405 410 415 Ile Tyr Ser Leu GlyGly Val Ser Ser Leu Glu Leu Thr Ala Ile Gln 420 425 430 Cys Lys Asn IlePro Asp Tyr Leu Asn Asp Arg Ala Ile Leu Glu Lys 435 440 445 His Phe SerLys Ile Ala Lys Val Gln Arg Val Phe Thr Arg Arg Ser 450 455 460 Lys LysLeu Ala Val Ile His Phe Phe Asp His Ala Ser Ala Ala Leu 465 470 475 480Ala Arg Lys Lys Gly Lys Gly Leu His Lys Asp Val Val Ile Phe Trp 485 490495 His Lys Lys Lys Ile Ser Pro Ser Lys Lys Leu Phe Pro Leu Lys Glu 500505 510 Lys Leu Gly Glu Ser Glu Ala Ser Gln Gly Ile Glu Asp Ser Pro Phe515 520 525 Gln His Ser Pro Leu Ser Lys Pro Ile Val Arg Pro Ala Ala GlySer 530 535 540 Leu Leu Ser Lys Ser Ser Pro Val Lys Lys Pro Ser Leu LeuLys Met 545 550 555 560 His Gln Phe Glu Ala Asp Pro Phe Asp Ser Gly SerGlu Gly Ser Glu 565 570 575 Gly Leu Gly Ser Cys Val Ser Ser Leu Ser ThrLeu Ile Gly Thr Val 580 585 590 Ala Asp Thr Ser Glu Glu Lys Tyr Arg LeuLeu Asp Gln Arg Asp Arg 595 600 605 Ile Met Arg Gln Ala Arg Val Lys ArgThr Asp Leu Asp Lys Ala Arg 610 615 620 Ala Phe Val Gly Thr Cys Pro AspMet Cys Pro Glu Lys Glu Arg Tyr 625 630 635 640 Leu Arg Glu Thr Arg SerGln Leu Ser Val Phe Glu Val Val Pro Gly 645 650 655 Thr Asp Gln Val AspHis Ala Ala Ala Val Lys Glu Tyr Ser Arg Ser 660 665 670 Ser Ala Asp GlnGlu Glu Pro Leu Pro His Glu Leu Arg Pro Ser Ala 675 680 685 Val Leu SerArg Thr Met Asp Tyr Leu Val Thr Gln Ile Met Asp Gln 690 695 700 Lys GluGly Ser Leu Arg Asp Trp Tyr Asp Phe Val Trp Asn Arg Thr 705 710 715 720Arg Gly Ile Arg Lys Asp Ile Thr Gln Gln His Leu Cys Asp Pro Leu 725 730735 Thr Val Ser Leu Ile Glu Lys Cys Thr Arg Phe His Ile His Cys Ala 740745 750 His Phe Met Cys Glu Glu Pro Met Ser Ser Phe Asp Ala Lys Ile Asn755 760 765 Asn Glu Asn Met Thr Lys Cys Leu Gln Ser Leu Lys Glu Met TyrGln 770 775 780 Asp Leu Arg Asn Lys Gly Val Phe Cys Ala Ser Glu Ala GluPhe Gln 785 790 795 800 Gly Tyr Asn Val Leu Leu Asn Leu Asn Lys Gly AspIle Leu Arg Glu 805 810 815 Val Gln Gln Phe His Pro Asp Val Arg Asn SerPro Glu Val Asn Phe 820 825 830 Ala Val Gln Ala Phe Ala Ala Leu Asn SerAsn Asn Phe Val Arg Phe 835 840 845 Phe Lys Leu Val Gln Ser Ala Ser TyrLeu Asn Ala Cys Leu Leu His 850 855 860 Cys Tyr Phe Asn Gln Ile Arg LysAsp Ala Leu Arg Ala Leu Asn Val 865 870 875 880 Ala Tyr Thr Val Ser ThrGln Arg Ser Thr Val Phe Pro Leu Asp Gly 885 890 895 Val Val Arg Met LeuLeu Phe Arg Asp Ser Glu Glu Ala Thr Asn Phe 900 905 910 Leu Asn Tyr HisGly Leu Thr Val Ala Asp Gly Cys Val Glu Leu Asn 915 920 925 Arg Ser AlaPhe Leu Glu Pro Glu Gly Leu Cys Lys Ala Arg Lys Ser 930 935 940 Val PheIle Gly Arg Lys Leu Thr Val Ser Val Gly Glu Val Val Asn 945 950 955 960Gly Gly Pro Leu Pro Pro Val Pro Arg His Thr Pro Val Cys Ser Phe 965 970975 Asn Ser Gln Asn Lys Tyr Val Gly Glu Ser Leu Ala Thr Glu Leu Pro 980985 990 Ile Ser Thr Gln Arg Ala Gly Gly Asp Pro Ala Gly Gly Gly Arg Gly995 1000 1005 Glu Asp Cys Glu Ala Glu Val Asp Leu Pro Thr Leu Ala ValLeu 1010 1015 1020 Pro Gln Pro Pro Pro Ala Ser Ser Ala Thr Pro Ala LeuHis Val 1025 1030 1035 Gln Pro Leu Ala Pro Ala Ala Ala Pro Ser Leu LeuGln Ala Ser 1040 1045 1050 Thr Gln Pro Glu Val Leu Leu Pro Lys Pro AlaPro Val Tyr Ser 1055 1060 1065 Asp Ser Asp Leu Val Gln Val Val Asp GluLeu Ile Gln Glu Ala 1070 1075 1080 Leu Gln Val Asp Cys Glu Glu Val SerSer Ala Gly Ala Ala Tyr 1085 1090 1095 Val Ala Ala Ala Leu Gly Val SerAsn Ala Ala Val Glu Asp Leu 1100 1105 1110 Ile Thr Ala Ala Thr Thr GlyIle Leu Arg His Val Ala Ala Glu 1115 1120 1125 Glu Val Ser Met Glu ArgGln Arg Leu Glu Glu Glu Lys Gln Arg 1130 1135 1140 Ala Glu Glu Glu ArgLeu Lys Gln Glu Arg Glu Leu Met Leu Thr 1145 1150 1155 Gln Leu Ser GluGly Leu Ala Ala Glu Leu Thr Glu Leu Thr Val 1160 1165 1170 Thr Glu CysVal Trp Glu Thr Cys Ser Gln Glu Leu Gln Ser Ala 1175 1180 1185 Val LysIle Asp Gln Lys Val Arg Val Ala Arg Cys Cys Glu Ala 1190 1195 1200 ValCys Ala His Leu Val Asp Leu Phe Leu Ala Glu Glu Ile Phe 1205 1210 1215Gln Thr Ala Lys Glu Thr Leu Gln Glu Leu Gln Cys Phe Cys Lys 1220 12251230 Tyr Leu Gln Arg Trp Arg Glu Ala Val Ala Ala Arg Lys Lys Phe 12351240 1245 Arg Arg Gln Met Arg Ala Phe Pro Ala Ala Pro Cys Cys Val Asp1250 1255 1260 Val Asn Asp Arg Leu Gln Ala Leu Val Pro Ser Ala Glu CysPro 1265 1270 1275 Ile Thr Glu Glu Asn Leu Ala Lys Gly Leu Leu Asp LeuGly His 1280 1285 1290 Ala Gly Lys Val Gly Val Ser Cys Thr Arg Leu ArgArg Leu Arg 1295 1300 1305 Asn Lys Thr Ala His Gln Ile Lys Val Gln HisPhe His Gln Gln 1310 1315 1320 Leu Leu Arg Asn Ala Ala Trp Ala Pro LeuAsp Leu Pro Ser Ile 1325 1330 1335 Val Ser Glu His Leu Pro Met Lys GlnLys Arg Arg Phe Trp Lys 1340 1345 1350 Leu Val Leu Val Leu Pro Asp ValGlu Glu Gln Thr Pro Glu Ser 1355 1360 1365 Pro Gly Arg Ile Leu Glu AsnTrp Leu Lys Val Lys Phe Thr Gly 1370 1375 1380 Asp Asp Ser Met Val GlyAsp Ile Gly Asp Asn Ala Gly Asp Ile 1385 1390 1395 Gln Thr Leu Ser ValPhe Asn Thr Leu Ser Ser Lys Gly Asp Gln 1400 1405 1410 Thr Val Ser ValAsn Val Cys Ile Lys Val Ala His Gly Thr Leu 1415 1420 1425 Ser Asp SerAla Leu Asp Ala Val Glu Thr Gln Lys Asp Leu Leu 1430 1435 1440 Gly ThrSer Gly Leu Met Leu Leu Leu Pro Pro Lys Val Lys Ser 1445 1450 1455 GluGlu Val Ala Glu Glu Glu Leu Ser Trp Leu Ser Ala Leu Leu 1460 1465 1470Gln Leu Lys Gln Leu Leu Gln Ala Lys Pro Phe Gln Pro Ala Leu 1475 14801485 Pro Leu Val Val Leu Val Pro Ser Ser Arg Gly Asp Ser Ala Gly 14901495 1500 Arg Ala Val Glu Asp Gly Leu Met Leu Gln Asp Leu Val Ser Ala1505 1510 1515 Lys Leu Ile Ser Asp Tyr Ile Val Val Glu Ile Pro Asp SerVal 1520 1525 1530 Asn Asp Leu Gln Gly Thr Val Lys Val Ser Gly Ala ValGln Trp 1535 1540 1545 Leu Ile Ser Gly Cys Pro Gln Ala Leu Asp Leu CysCys Gln Thr 1550 1555 1560 Leu Val Gln Tyr Val Glu Asp Gly Ile Ser ArgGlu Phe Ser Arg 1565 1570 1575 Arg Phe Phe His Asp Arg Arg Glu Arg ArgLeu Ala Ser Leu Pro 1580 1585 1590 Ser Gln Glu Pro Ser Thr Ile Ile GluLeu Phe Asn Ser Val Leu 1595 1600 1605 Gln Phe Leu Ala Ser Val Val SerSer Glu Gln Leu Cys Asp Ile 1610 1615 1620 Ser Trp Pro Val Met Glu PheAla Glu Val Gly Gly Ser Gln Leu 1625 1630 1635 Leu Pro His Leu His TrpAsn Ser Pro Glu His Leu Ala Trp Leu 1640 1645 1650 Lys Gln Ala Val LeuGly Phe Gln Leu Pro Gln Met Asp Leu Pro 1655 1660 1665 Pro Pro Gly AlaPro Trp Leu Pro Val Cys Ser Met Val Ile Gln 1670 1675 1680 Tyr Thr SerGln Ile Pro Ser Ser Ser Gln Thr Gln Pro Val Leu 1685 1690 1695 Gln SerGln Ala Glu Asn Leu Leu Cys Arg Thr Tyr Gln Lys Trp 1700 1705 1710 LysAsn Lys Ser Leu Ser Pro Gly Gln Glu Leu Gly Pro Ser Val 1715 1720 1725Ala Glu Ile Pro Trp Asp Asp Ile Ile Thr Leu Cys Ile Asn His 1730 17351740 Lys Leu Arg Asp Trp Thr Pro Pro Arg Leu Pro Val Thr Leu Glu 17451750 1755 Ala Leu Ser Glu Asp Gly Gln Ile Cys Val Tyr Phe Phe Lys Asn1760 1765 1770 Leu Leu Arg Lys Tyr His Val Pro Ser Ser Trp Glu Gln AlaArg 1775 1780 1785 Met Gln Thr Gln Arg Glu Leu Gln Leu Ser His Gly ArgSer Gly 1790 1795 1800 Met Arg Ser Ile His Pro Pro Thr Ser Thr Phe ProThr Pro Leu 1805 1810 1815 Leu His Val His Gln Lys Gly Lys Lys Lys GluGlu Ser Gly Arg 1820 1825 1830 Glu Gly Ser Leu Ser Thr Glu Asp Leu LeuArg Gly Ala Ser Ala 1835 1840 1845 Glu Glu Leu Leu Ala Gln Ser Leu SerSer Ser Leu Leu Glu Glu 1850 1855 1860 Lys Glu Glu Asn Lys Arg Phe GluAsp Gln Leu Gln Gln Trp Leu 1865 1870 1875 Ser Gln Asp Ser Gln Ala PheThr Glu Ser Thr Arg Leu Pro Leu 1880 1885 1890 Tyr Leu Pro Gln Thr LeuVal Ser Phe Pro Asp Ser Ile Lys Thr 1895 1900 1905 Gln Thr Met Val LysThr Ser Thr Ser Pro Gln Asn Ser Gly Thr 1910 1915 1920 Gly Lys Gln LeuArg Phe Ser Glu Ala Ser Gly Ser Ser Leu Thr 1925 1930 1935 Glu Lys LeuLys Leu Leu Glu Arg Leu Ile Gln Ser Ser Arg Ala 1940 1945 1950 Glu GluAla Ala Ser Glu Leu His Leu Ser Ala Leu Leu Glu Met 1955 1960 1965 ValAsp Met 1970 2 6429 DNA Mouse 2 gttgcggtgc ggtgggcccg gtagaggctgcacgcagact gtgggcgagc acaagcgctg 60 gcgacagtgg ccgtatctgg cggacttgctcctccctccg cggcctccgc tgtcccttgt 120 gtctttgccg agttgctgaa ggccttcactagtcttcgct cgaaggcgtc tgttaaccta 180 gcggccggct tccggagtgt taagcatcggggataaaaag ctattatttc tagaccaggg 240 catcgcaagt tcgagttacc gggagaaaaatgagatggtc atcctgagga tgaaggagag 300 cttcccctgg caacagataa tttaaagaggagagctactt gtgtatagtc catatttatt 360 gccttcagat aattggcttg aagatgcacccggtgaaccc cttcggaggc agcagcccaa 420 gtgcttttgc ggtatcttcc agcaccacgggaacatatca gactaaatca ccatttcgat 480 ttggccagcc ttcccttttt ggacagaacagcacacccag caagagcctg gcgttttcac 540 aagtaccaag ctttgcaaca ccctctggaggaagccattc ttcctccttg ccagcatttg 600 gactcaccca aacctcaagt gtgggactcttctctagtct cgaatccaca ccttctttcg 660 cagctacttc gagttcctct gtgcccggcaatacggcatt cagctttaag tcaacctcta 720 gcgttggggt tttcccaagt ggcgctacttttgggccaga aaccggagaa gtagcaggtt 780 ctggctttcg gaagacggaa ttcaagtttaaacctctgga aaatgcagtc ttcaaaccga 840 taccggggcc tgagtcagag ccagaaaaaacccagagcca gatttcttct ggatttttta 900 cattttccca tcccgttggt agcgggtctggaggcctgac ccctttttct ttcccacagg 960 tgacaaatag ttcggtgact agctcaagttttatcttttc gaaaccagtt actagtaata 1020 ctcctgcctt tgcctctcct ttgtctaaccaaaatgtaga agaagagaag agggtttcta 1080 cgtcagcgtt tggaagctca aacagtagcttcagtacttt ccccacagcg tcaccaggat 1140 ctttggggga gcccttccca gctaacaaaccaagcctccg ccaaggatgt gaggaagcca 1200 tctcccaggt ggagccactt cccaccctcatgaagggatt aaagaggaaa gaggaccagg 1260 atcgctcccc gaggagacat tgccacgaggcagcagaaga ccctgatccc ctgtccaggg 1320 gcgaccatcc cccagataaa cggccagtccgcctcaacag accccgggga ggtactttgt 1380 ttggccggac aatacaggag gtcttcaaaagcaataaaga ggcaggccgc ctgggcagca 1440 aggaatccaa ggagagtggc tttgcggaacctggggaaag tgaccacgcg gccgtcccag 1500 gagggagtca gtccaccatg gtaccttcccgccttccagc tgtgactaaa gaggaagaag 1560 aaagtagaga tgagaaagaa gattctctcaggggaaagtc tgtgcgccag agtaagcgaa 1620 gggaagagtg gatctacagc ctcgggggcgtgtcttcttt agagctcaca gccatccagt 1680 gcaagaacat ccccgactac ctcaacgacagagccatcct ggagaaacac ttcagcaaaa 1740 tcgctaaagt ccagcgggtc ttcaccagacgcagcaagaa gctcgccgtg attcattttt 1800 tcgaccacgc atcggcagcc ctggctaggaagaaggggaa aggtctgcat aaggacgtgg 1860 ttatcttttg gcacaagaag aaaataagtcccagcaagaa actctttccc ctgaaggaga 1920 agcttggtga gagtgaagcc agccagggcatcgaggactc cccctttcag cactcgcctc 1980 tcagcaagcc catcgtgagg cctgcagccggcagcctcct cagcaaaagc tctccagtga 2040 agaagccgag tcttctgaag atgcaccagtttgaggcgga tccttttgac tctggatctg 2100 agggctccga gggccttggt tcttgcgtgtcatctcttag caccctgata gggactgtgg 2160 cagacacatc tgaggagaag taccgccttctggaccagag agaccgcatc atgcggcaag 2220 ctcgagtgaa gaggacggac ctggacaaagccagggcatt tgttgggact tgccctgaca 2280 tgtgtcccga gaaggagcgg tacttgagggagacccggag ccagctgagc gtgtttgaag 2340 ttgtcccagg gactgaccag gtggaccatgcagcagccgt gaaggagtac agccggtcct 2400 ctgcagatca ggaggagccc ctgccacatgagctgagacc ctcagcagtt ctcagcagga 2460 ccatggacta cctggtgacc cagatcatggaccaaaagga aggcagcctt cgggattggt 2520 atgacttcgt gtggaaccgc acccggggtatacggaagga cataacacag cagcacctct 2580 gtgatcccct gacggtgtct ctgatcgagaagtgtacccg atttcacatt cactgtgccc 2640 actttatgtg tgaggagcct atgtcttcctttgatgccaa gatcaacaat gagaacatga 2700 ccaagtgtct acagagtctg aaggagatgtaccaggacct gaggaacaag ggtgtttttt 2760 gtgccagtga agcagagttt cagggctacaatgtcctgct taatctcaac aaaggagaca 2820 ttttgagaga agtgcagcag ttccaccctgacgttaggaa ctccccagag gtgaacttcg 2880 ctgtccaggc ttttgctgca ttgaacagcaataattttgt gagatttttc aaactggttc 2940 agtcagcttc ttacctgaat gcgtgcctgttacactgtta ctttaatcag atccgcaagg 3000 atgccctccg ggcactcaat gttgcttatactgtaagcac acagcgctct accgtcttcc 3060 ccctggatgg tgtcgtccgc atgctgctgttcagagatag tgaagaggcg acaaacttcc 3120 tcaattacca tggcctcact gtagctgatggctgtgttga gctgaatcgg tcggcattct 3180 tggaaccgga gggattatgc aaggccaggaagtcagtgtt tattggccgg aagctgacgg 3240 tgtcagttgg ggaagttgtg aatggagggccgttgccccc tgttcctcgc catacacctg 3300 tgtgcagctt caactcccag aataagtacgttggagagag cctggctacg gagctgccca 3360 tcagcactca gagagctggt ggagacccagcaggtggtgg cagaggagag gactgtgagg 3420 cagaggtgga cttgccaaca ttggcggtcctcccacagcc gcctcctgca tcctcagcca 3480 cgccggcgct tcatgtccag ccactggccccagccgcagc acccagcctt ctccaggcct 3540 ccacgcagcc tgaggtgctg cttccaaagcctgcgcctgt gtactctgac tcggacctgg 3600 tacaggtggt ggacgagctc atccaggaggctctgcaagt ggactgtgag gaagtcagct 3660 ccgctggggc agcctacgta gccgcagctctgggcgtttc caatgctgct gtggaggatc 3720 tgattactgc tgcgaccacg ggcattctgaggcacgttgc cgctgaggaa gtttccatgg 3780 aaaggcagag actagaggaa gagaagcaacgagctgagga ggaacggttg aagcaagaga 3840 gagaactgat gttaactcag ctgagcgagggtctggccgc agagctgaca gaactcacgg 3900 tgacagagtg tgtgtgggaa acctgctctcaggagctaca gagtgcagta aaaatagacc 3960 agaaggtccg tgtggcccgc tgttgtgaagccgtctgtgc acacctggtg gatttgtttc 4020 ttgctgagga aattttccag actgcaaaagagacactcca ggaactccag tgtttctgca 4080 agtatctaca acggtggagg gaggctgttgcagctcggaa gaaattccgg cgtcagatgc 4140 gggccttccc tgcagcgcca tgctgtgtggatgtgaatga ccggctgcag gcactagtgc 4200 ccagcgcaga gtgccccatt actgaggagaacctggccaa gggtcttttg gacctgggcc 4260 acgcaggcaa agtaggcgtc tcctgtaccaggttgaggcg gcttagaaac aagacagctc 4320 accagataaa ggtccagcac ttccaccagcagctgctgag gaatgctgca tgggcacctc 4380 tggacctgcc atccattgtg tctgagcacctccccatgaa gcagaagcga aggttttgga 4440 aactggtgct ggtgttgcct gatgtggaagagcagactcc agagagtcct ggcagaatac 4500 tagaaaactg gctaaaggtc aaattcacaggagatgacag catggtgggt gacataggag 4560 ataatgctgg tgatatccag accctctcagtctttaatac acttagtagt aaaggggatc 4620 aaacagtttc tgtcaacgtg tgtataaaggtggctcatgg cacccttagt gacagtgccc 4680 ttgatgctgt ggagacccag aaggacctgttgggaaccag tgggctcatg ctgctgcttc 4740 ccccgaaagt gaagagtgag gaggtggcagaggaggaact gtcctggctg tcggctttac 4800 tgcagctcaa gcagcttctg caggccaagcccttccagcc tgccctgccg ctggtggtcc 4860 tcgtgcccag ctccagaggg gactccgcggggagggcagt agaggacggt ctgatgttac 4920 aggatttggt ttcagccaag ctgatttccgattacattgt tgttgagatt cctgactctg 4980 ttaatgattt acaaggcaca gtgaaggtttctggagcagt ccagtggctg atctccggat 5040 gtcctcaagc cctagacctt tgctgccagacccttgttca gtatgttgag gatgggatca 5100 gccgcgagtt cagccgtcgg tttttccacgacaggagaga gaggcgcctg gctagcctgc 5160 cctcccagga gcctagcacc attattgagttgttcaacag tgtgctgcag ttcctggcct 5220 ctgtggtatc ctctgagcag ctgtgtgacatctcctggcc tgtcatggaa tttgccgaag 5280 tgggaggcag ccagctgctt cctcacctgcactggaactc accagagcat ctagcgtggc 5340 tgaaacaagc tgtgcttggg ttccagcttccacagatgga ccttccaccc ccaggggccc 5400 cctggctccc tgtgtgttcc atggtcattcagtacacctc ccagattccc agctcaagcc 5460 agacacagcc tgtcctccag tcccaggcggagaacctgct gtgcagaaca taccagaagt 5520 ggaagaacaa gagcctctct ccaggccaggagttggggcc ttctgttgcc gagatcccgt 5580 gggatgacat catcacctta tgcatcaatcataagctgag ggactggaca ccccccaggc 5640 tccctgtcac attagaggcg ctgagtgaagatggtcaaat atgtgtgtat tttttcaaaa 5700 accttttaag aaaataccac gttccctcgtcatgggaaca ggccagaatg cagacgcagc 5760 gggaactgca gctgagtcat ggacgttcggggatgaggtc catccatcct cctacaagca 5820 cttttcctac tccattgctt catgtacaccagaaagggaa gaaaaaggaa gagagtggcc 5880 gagaggggag cctcagtaca gaggacctcctgcggggggc ttctgcagaa gagctcctgg 5940 cacagagtct gtccagcagt cttctggaagagaaggaaga gaacaagagg tttgaagatc 6000 aacttcagca gtggttatcg caagactcacaggcattcac agagtcaact cggcttcctc 6060 tctacctccc tcagacgcta gtgtcctttcctgattctat caaaactcag accatggtga 6120 aaacatctac aagtcctcag aattcaggaacaggaaagca gttgaggttc tcagaggcat 6180 ccggttcatc cctgacggaa aagctgaagctcctggaaag gctgatccag agctcaaggg 6240 cggaagaagc agcctccgag ctgcacctctctgcactgct ggagatggtg gacatgtagc 6300 tgtctgacgg gagacggatc tctaattcataatgctttgt ctgtattcaa ttgtgttata 6360 gatgctgttg gaaatgtgac tattaattatgcaaataaac tttttgaatc attccaaaaa 6420 aaaaaccat 6429 3 1980 PRT Homosapiens 3 Met Asn Pro Thr Asn Pro Phe Ser Gly Gln Gln Pro Ser Ala PheSer 1 5 10 15 Ala Ser Ser Ser Asn Val Gly Thr Leu Pro Ser Lys Pro ProPhe Arg 20 25 30 Phe Gly Gln Pro Ser Leu Phe Gly Gln Asn Ser Thr Leu SerGly Lys 35 40 45 Ser Ser Gly Phe Ser Gln Val Ser Ser Phe Pro Ala Ser SerGly Val 50 55 60 Ser His Ser Ser Ser Val Gln Thr Leu Gly Phe Thr Gln ThrSer Ser 65 70 75 80 Val Gly Pro Phe Ser Gly Leu Glu His Thr Ser Thr PheVal Ala Thr 85 90 95 Ser Gly Pro Ser Ser Ser Ser Val Leu Gly Asn Thr GlyPhe Ser Phe 100 105 110 Lys Ser Pro Thr Ser Val Gly Ala Phe Pro Ser ThrSer Ala Phe Gly 115 120 125 Gln Glu Ala Gly Glu Ile Val Asn Ser Gly PheGly Lys Thr Glu Phe 130 135 140 Ser Phe Lys Pro Leu Glu Asn Ala Val PheLys Pro Ile Leu Gly Ala 145 150 155 160 Glu Ser Glu Pro Glu Lys Thr GlnSer Gln Ile Ala Ser Gly Phe Phe 165 170 175 Thr Phe Ser His Pro Ile SerSer Ala Pro Gly Gly Leu Ala Pro Phe 180 185 190 Ser Phe Pro Gln Val ThrSer Ser Ser Ala Thr Thr Ser Asn Phe Thr 195 200 205 Phe Ser Lys Pro ValSer Ser Asn Asn Ser Leu Ser Ala Phe Thr Pro 210 215 220 Ala Leu Ser AsnGln Asn Val Glu Glu Glu Lys Arg Gly Pro Lys Ser 225 230 235 240 Ile PheGly Ser Ser Asn Asn Ser Phe Ser Ser Phe Pro Val Ser Ser 245 250 255 AlaVal Leu Gly Glu Pro Phe Gln Ala Ser Lys Ala Gly Val Arg Gln 260 265 270Gly Cys Glu Glu Ala Val Ser Gln Val Glu Pro Leu Pro Ser Leu Met 275 280285 Lys Gly Leu Lys Arg Lys Glu Asp Gln Asp Arg Ser Pro Arg Arg His 290295 300 Gly His Glu Pro Ala Glu Asp Ser Asp Pro Leu Ser Arg Gly Asp His305 310 315 320 Pro Pro Asp Lys Arg Pro Val Arg Leu Asn Arg Pro Arg GlyGly Thr 325 330 335 Leu Phe Gly Arg Thr Ile Gln Asp Val Phe Lys Ser AsnLys Glu Val 340 345 350 Gly Arg Leu Gly Asn Lys Glu Ala Lys Lys Glu ThrGly Phe Val Glu 355 360 365 Ser Ala Glu Ser Asp His Met Ala Ile Pro GlyGly Asn Gln Ser Val 370 375 380 Leu Ala Pro Ser Arg Ile Pro Gly Val AsnLys Glu Glu Glu Thr Glu 385 390 395 400 Ser Arg Glu Lys Lys Glu Asp SerLeu Arg Gly Thr Pro Ala Arg Gln 405 410 415 Ser Asn Arg Ser Glu Ser ThrAsp Ser Leu Gly Gly Leu Ser Pro Ser 420 425 430 Glu Val Thr Ala Ile GlnCys Lys Asn Ile Pro Asp Tyr Leu Asn Asp 435 440 445 Arg Thr Ile Leu GluAsn His Phe Gly Lys Ile Ala Lys Val Gln Arg 450 455 460 Ile Phe Thr ArgArg Ser Lys Lys Leu Ala Val Val His Phe Phe Asp 465 470 475 480 His AlaSer Ala Ala Leu Ala Arg Lys Lys Gly Lys Ser Leu His Lys 485 490 495 AspMet Ala Ile Phe Trp His Arg Lys Lys Ile Ser Pro Asn Lys Lys 500 505 510Pro Phe Ser Leu Lys Glu Lys Lys Pro Gly Asp Gly Glu Val Ser Pro 515 520525 Ser Thr Glu Asp Ala Pro Phe Gln His Ser Pro Leu Gly Lys Ala Ala 530535 540 Gly Arg Thr Gly Ala Ser Ser Leu Leu Asn Lys Ser Ser Pro Val Lys545 550 555 560 Lys Pro Ser Leu Leu Lys Ala His Gln Phe Glu Gly Asp SerPhe Asp 565 570 575 Ser Ala Ser Glu Gly Ser Glu Gly Leu Gly Pro Cys ValLeu Ser Leu 580 585 590 Ser Thr Leu Ile Gly Thr Val Ala Glu Thr Ser LysGlu Lys Tyr Arg 595 600 605 Leu Leu Asp Gln Arg Asp Arg Ile Met Arg GlnAla Arg Val Lys Arg 610 615 620 Thr Asp Leu Asp Lys Ala Arg Thr Phe ValGly Thr Cys Leu Asp Met 625 630 635 640 Cys Pro Glu Lys Glu Arg Tyr MetArg Glu Thr Arg Ser Gln Leu Ser 645 650 655 Val Phe Glu Val Val Pro GlyThr Asp Gln Val Asp His Ala Ala Ala 660 665 670 Val Lys Glu Tyr Ser ArgSer Ser Ala Asp Gln Glu Glu Pro Leu Pro 675 680 685 His Glu Leu Arg ProLeu Pro Val Leu Ser Arg Thr Met Asp Tyr Leu 690 695 700 Val Thr Gln IleMet Asp Gln Lys Glu Gly Ser Leu Arg Asp Trp Tyr 705 710 715 720 Asp PheVal Trp Asn Arg Thr Arg Gly Ile Arg Lys Asp Ile Thr Gln 725 730 735 GlnHis Leu Cys Asp Pro Leu Thr Val Ser Leu Ile Glu Lys Cys Thr 740 745 750Arg Phe His Ile His Cys Ala His Phe Met Cys Glu Glu Pro Met Ser 755 760765 Ser Phe Asp Ala Lys Ile Asn Asn Glu Asn Met Thr Lys Cys Leu Gln 770775 780 Ser Leu Lys Glu Met Tyr Gln Asp Leu Arg Asn Lys Gly Val Phe Cys785 790 795 800 Ala Ser Glu Ala Glu Phe Gln Gly Tyr Asn Val Leu Leu SerLeu Asn 805 810 815 Lys Gly Asp Ile Leu Arg Glu Val Gln Gln Phe His ProAla Val Arg 820 825 830 Asn Ser Ser Glu Val Lys Phe Ala Val Gln Ala PheAla Ala Leu Asn 835 840 845 Ser Asn Asn Phe Val Arg Phe Phe Lys Leu ValGln Ser Ala Ser Tyr 850 855 860 Leu Asn Ala Cys Leu Leu His Cys Tyr PheSer Gln Ile Arg Lys Asp 865 870 875 880 Ala Leu Arg Ala Leu Asn Phe AlaTyr Thr Val Ser Thr Gln Arg Ser 885 890 895 Thr Ile Phe Pro Leu Asp GlyVal Val Arg Met Leu Leu Phe Arg Asp 900 905 910 Cys Glu Glu Ala Thr AspPhe Leu Thr Cys His Gly Leu Thr Val Ser 915 920 925 Asp Gly Cys Val GluLeu Asn Arg Ser Ala Phe Leu Glu Pro Glu Gly 930 935 940 Leu Ser Lys ThrArg Lys Ser Val Phe Ile Thr Arg Lys Leu Thr Val 945 950 955 960 Ser ValGly Glu Ile Val Asn Gly Gly Pro Leu Pro Pro Val Pro Arg 965 970 975 HisThr Pro Val Cys Ser Phe Asn Ser Gln Asn Lys Tyr Ile Gly Glu 980 985 990Ser Leu Ala Ala Glu Leu Pro Val Ser Thr Gln Arg Pro Gly Ser Asp 995 10001005 Thr Val Gly Gly Gly Arg Gly Glu Glu Cys Gly Val Glu Pro Asp 10101015 1020 Ala Pro Leu Ser Ser Leu Pro Gln Ser Leu Pro Ala Pro Ala Pro1025 1030 1035 Ser Pro Val Pro Leu Pro Pro Val Leu Ala Leu Thr Pro SerVal 1040 1045 1050 Ala Pro Ser Leu Phe Gln Leu Ser Val Gln Pro Glu ProPro Pro 1055 1060 1065 Pro Glu Pro Val Pro Met Tyr Ser Asp Glu Asp LeuAla Gln Val 1070 1075 1080 Val Asp Glu Leu Ile Gln Glu Ala Leu Gln ArgAsp Cys Glu Glu 1085 1090 1095 Val Gly Ser Ala Gly Ala Ala Tyr Ala AlaAla Ala Leu Gly Val 1100 1105 1110 Ser Asn Ala Ala Met Glu Asp Leu LeuThr Ala Ala Thr Thr Gly 1115 1120 1125 Ile Leu Arg His Ile Ala Ala GluGlu Val Ser Lys Glu Arg Glu 1130 1135 1140 Arg Arg Glu Gln Glu Arg GlnArg Ala Glu Glu Glu Arg Leu Lys 1145 1150 1155 Gln Glu Arg Glu Leu ValLeu Ser Glu Leu Ser Gln Gly Leu Ala 1160 1165 1170 Val Glu Leu Met GluArg Val Met Met Glu Phe Val Arg Glu Thr 1175 1180 1185 Cys Ser Gln GluLeu Lys Asn Ala Val Glu Thr Asp Gln Arg Val 1190 1195 1200 Arg Val AlaArg Cys Cys Glu Asp Val Cys Ala His Leu Val Asp 1205 1210 1215 Leu PheLeu Val Glu Glu Ile Phe Gln Thr Ala Lys Glu Thr Leu 1220 1225 1230 GlnGlu Leu Gln Cys Phe Cys Lys Tyr Leu Gln Arg Trp Arg Glu 1235 1240 1245Ala Val Thr Ala Arg Lys Lys Leu Arg Arg Gln Met Arg Ala Phe 1250 12551260 Pro Ala Ala Pro Cys Cys Val Asp Val Ser Asp Arg Leu Arg Ala 12651270 1275 Leu Ala Pro Ser Ala Glu Cys Pro Ile Ala Glu Glu Asn Leu Ala1280 1285 1290 Arg Gly Leu Leu Asp Leu Gly His Ala Gly Arg Leu Gly IleSer 1295 1300 1305 Cys Thr Arg Leu Arg Arg Leu Arg Asn Lys Thr Ala HisGln Met 1310 1315 1320 Lys Val Gln His Phe Tyr Gln Gln Leu Leu Ser AspVal Ala Trp 1325 1330 1335 Ala Ser Leu Asp Leu Pro Ser Leu Val Ala GluHis Leu Pro Gly 1340 1345 1350 Arg Gln Glu His Val Phe Trp Lys Leu ValLeu Val Leu Pro Asp 1355 1360 1365 Val Glu Glu Gln Ser Pro Glu Ser CysGly Arg Ile Leu Ala Asn 1370 1375 1380 Trp Leu Lys Val Lys Phe Met GlyAsp Glu Gly Ser Val Asp Asp 1385 1390 1395 Thr Ser Ser Asp Ala Gly GlyIle Gln Thr Leu Ser Leu Phe Asn 1400 1405 1410 Ser Leu Ser Ser Lys GlyAsp Gln Met Ile Ser Val Asn Val Cys 1415 1420 1425 Ile Lys Val Ala HisGly Ala Leu Ser Asp Gly Ala Ile Asp Ala 1430 1435 1440 Val Glu Thr GlnLys Asp Leu Leu Gly Ala Ser Gly Leu Met Leu 1445 1450 1455 Leu Leu ProPro Lys Met Lys Ser Glu Asp Met Ala Glu Glu Asp 1460 1465 1470 Val TyrTrp Leu Ser Ala Leu Leu Gln Leu Lys Gln Leu Leu Gln 1475 1480 1485 AlaLys Pro Phe Gln Pro Ala Leu Pro Leu Val Val Leu Val Pro 1490 1495 1500Ser Pro Gly Gly Asp Ala Val Glu Lys Glu Val Glu Asp Gly Leu 1505 15101515 Met Leu Gln Asp Leu Val Ser Ala Lys Leu Ile Ser Asp Tyr Thr 15201525 1530 Val Thr Glu Ile Pro Asp Thr Ile Asn Asp Leu Gln Gly Ser Thr1535 1540 1545 Lys Val Leu Gln Ala Val Gln Trp Leu Val Ser His Cys ProHis 1550 1555 1560 Ser Leu Asp Leu Cys Cys Gln Thr Leu Ile Gln Tyr ValGlu Asp 1565 1570 1575 Gly Ile Gly His Glu Phe Ser Gly Arg Phe Phe HisAsp Arg Arg 1580 1585 1590 Glu Arg Arg Leu Gly Gly Leu Ala Ser Gln GluPro Gly Ala Ile 1595 1600 1605 Ile Glu Leu Phe Asn Ser Val Leu Gln PheLeu Ala Ser Val Val 1610 1615 1620 Ser Ser Glu Gln Leu Cys Asp Leu SerTrp Pro Val Thr Glu Phe 1625 1630 1635 Ala Glu Ala Gly Gly Ser Arg LeuLeu Pro His Leu His Trp Asn 1640 1645 1650 Ala Pro Glu His Leu Ala TrpLeu Lys Gln Ala Val Leu Gly Phe 1655 1660 1665 Gln Leu Pro Gln Met AspLeu Pro Pro Leu Gly Ala Pro Trp Leu 1670 1675 1680 Pro Val Cys Ser MetVal Val Gln Tyr Ala Ser Gln Ile Pro Ser 1685 1690 1695 Ser Arg Gln ThrGln Pro Val Leu Gln Ser Gln Val Glu Asn Leu 1700 1705 1710 Leu His ArgThr Tyr Cys Arg Trp Lys Ser Lys Ser Pro Ser Pro 1715 1720 1725 Val HisGly Ala Gly Pro Ser Val Met Glu Ile Pro Trp Asp Asp 1730 1735 1740 LeuIle Ala Leu Cys Ile Asn His Lys Leu Arg Asp Trp Thr Pro 1745 1750 1755Pro Arg Leu Pro Val Thr Ser Glu Ala Leu Ser Glu Asp Gly Gln 1760 17651770 Ile Cys Val Tyr Phe Phe Lys Asn Asp Leu Lys Lys Tyr Asp Val 17751780 1785 Pro Leu Ser Trp Glu Gln Ala Arg Leu Gln Thr Gln Lys Glu Leu1790 1795 1800 Gln Leu Arg Glu Gly Arg Leu Ala Ile Lys Pro Phe His ProSer 1805 1810 1815 Ala Asn Asn Phe Pro Ile Pro Leu Leu His Met His ArgAsn Trp 1820 1825 1830 Lys Arg Ser Thr Glu Cys Ala Gln Glu Gly Arg IlePro Ser Thr 1835 1840 1845 Glu Asp Leu Met Arg Gly Ala Ser Ala Glu GluLeu Leu Ala Gln 1850 1855 1860 Cys Leu Ser Ser Ser Leu Leu Leu Glu LysGlu Glu Asn Lys Arg 1865 1870 1875 Phe Glu Asp Gln Leu Gln Gln Trp LeuSer Glu Asp Ser Gly Ala 1880 1885 1890 Phe Thr Asp Leu Thr Ser Leu ProLeu Tyr Leu Pro Gln Thr Leu 1895 1900 1905 Val Ser Leu Ser His Thr IleGlu Pro Val Met Lys Thr Ser Val 1910 1915 1920 Thr Thr Ser Pro Gln SerAsp Met Met Arg Glu Gln Leu Gln Leu 1925 1930 1935 Ser Glu Ala Thr GlyThr Cys Leu Gly Glu Arg Leu Lys His Leu 1940 1945 1950 Glu Arg Leu IleArg Ser Ser Arg Glu Glu Glu Val Ala Ser Glu 1955 1960 1965 Leu His LeuSer Ala Leu Leu Asp Met Val Asp Ile 1970 1975 1980 4 6114 DNA Homosapiens 4 gtaatactta attaccttct aataattgga gcagaagatg aacccaactaatcctttcag 60 tgggcagcag cctagtgctt tttcggcgtc ttctagtaat gtaggaacacttccatctaa 120 gccgccattt cgatttggtc aaccttctct ttttggacaa aacagtaccttatctgggaa 180 gagctcggga ttttcacagg tatccagctt tccagcgtct tctggagtaagtcattcctc 240 ttcagtgcaa acattagggt tcacccaaac ctcaagtgtt ggacccttttctggacttga 300 gcacacttcc acctttgtgg ctacctctgg gccttcaagt tcatctgtgctgggaaacac 360 aggatttagt tttaaatcac ccaccagtgt tggggctttc ccaagcacttctgcttttgg 420 acaagaagct ggagaaatag tgaactctgg ttttgggaaa acagaattcagctttaaacc 480 tctggaaaat gcagtgttca aaccaatact gggggctgaa tctgagccagagaaaaccca 540 gagccaaatt gcttctgggt tttttacatt ttcccaccca attagtagtgcacctggagg 600 cctggcccct ttctcttttc ctcaagtaac aagtagttca gctaccacttcaaattttac 660 cttttcaaaa cctgttagta gtaataattc attatctgcc tttacccctgctttgtcaaa 720 ccaaaatgta gaggaagaga agagaggacc taagtcaata tttggaagttctaataatag 780 cttcagtagc ttccctgtat catctgcggt tttgggcgaa cctttccaggctagcaaagc 840 aggtgtcagg caggggtgtg aagaagctgt ttcccaggtg gaaccacttcccagcctaat 900 gaaaggactg aaaaggaagg aggaccagga tcgctcccca aggagacatggccacgagcc 960 agcagaagat tcggatcctc tgtcccgggg cgatcatcct ccagacaaacgacctgtccg 1020 cctgaatcga ccccggggag gtactttatt tggtcggacg atacaggatgttttcaaaag 1080 caataaggaa gtaggtcgtc tgggcaacaa ggaggccaaa aaggaaactggctttgttga 1140 gtctgcagaa agtgaccaca tggctatccc aggagggaat cagtctgtcctggcaccttc 1200 ccggattcca ggtgtgaata aagaggaaga aactgaaagt agagagaagaaagaagattc 1260 tctaagagga actccggcgc gtcagagtaa cagaagcgag agcacagacagtcttggggg 1320 cttgtctccc tctgaagtca cagccatcca gtgcaagaac atccctgactacctcaacga 1380 caggaccatt ctggagaacc attttggcaa aattgctaaa gtgcagcgcatctttaccag 1440 gcgcagcaaa aagcttgcag tggtacattt ctttgatcat gcatctgcagccctggctag 1500 aaagaagggg aaaagtttgc ataaagacat ggctatcttt tggcacaggaagaaaataag 1560 ccccaataag aaaccctttt ccctgaagga gaagaaacca ggtgacggtgaagtcagccc 1620 gagcacagag gatgcaccct ttcagcactc tcctcttggc aaggccgcagggaggactgg 1680 tgctagcagc ctcctgaata aaagctctcc agtgaagaag ccaagtcttctaaaggccca 1740 ccaattcgag ggagactctt ttgactcagc ctccgagggc tccgagggcctcgggccatg 1800 tgtgctctcc ctcagtaccc tgataggcac tgtggctgag acatccaaggagaagtaccg 1860 cctgcttgac cagagagaca ggatcatgcg gcaagctcgg gtgaagagaaccgatctgga 1920 caaagcgagg acttttgttg gcacctgcct ggatatgtgt cctgagaaggagaggtacat 1980 gcgggagacc cgtagccagc tgagcgtgtt cgaagtggtc ccagggactgaccaggtgga 2040 ccacgcagca gctgtgaaag agtacagtcg gtcctcggcg gatcaggaggagcccctgcc 2100 ccacgagctg cggcccttgc cagtgctcag caggaccatg gactacctggtgacccagat 2160 catggaccag aaggagggca gcctgcggga ttggtatgac ttcgtgtggaaccgcacgcg 2220 tggcatacgg aaggatatca cgcagcagca cctctgtgac cccctgacggtgtccctgat 2280 tgagaagtgc acccggtttc acatccactg tgcccacttc atgtgtgaggagcccatgtc 2340 ctcctttgat gccaagatca ataatgagaa catgaccaag tgcctgcagagcctgaagga 2400 gatgtaccag gacctgagaa acaagggtgt cttctgtgcc agcgaagcggagttccaggg 2460 ctacaatgtt ctgctcagtc tcaacaaggg agacatccta agagaagtacaacagttcca 2520 tcctgctgtt agaaactcat ctgaggtgaa atttgctgtt caggcttttgctgcattgaa 2580 cagtaataat tttgtgagat ttttcaaact ggtccagtca gcttcttacctgaacgcttg 2640 tcttttacac tgttacttca gtcagatccg caaggatgct ctccgggcgctcaactttgc 2700 gtacacggtg agcacacagc gatctaccat ctttcccctg gatggtgtggtgcgcatgct 2760 gctgttcaga gactgtgaag aggccaccga cttcctcacc tgccacggcctcaccgtttc 2820 cgacggctgt gtggagctga accggtctgc attcctggaa ccagagggattatccaagac 2880 caggaagtcg gtgtttatta ctaggaagct gacggtgtca gtcggggaaattgtgaacgg 2940 agggccattg ccccccgtcc ctcgtcacac ccctgtgtgc agcttcaactcccagaacaa 3000 gtacatcggg gagagcctgg ccgcggagct gcccgtcagc acccagagacccggctccga 3060 cacagtgggc ggagggagag gagaggagtg tggtgtagag ccggatgcacccctgtccag 3120 tctcccacag tctctaccag cccctgcgcc ctcaccagtg cctctgcctcctgtcctggc 3180 actgaccccg tctgtggcgc ccagcctctt ccagctgtct gtgcagcctgaaccaccgcc 3240 tccagagccc gtgcccatgt actctgacga ggacctggcg caggtggtggacgagctcat 3300 ccaggaggcc ctgcagaggg actgtgagga agttggctct gcgggtgctgcctacgcagc 3360 tgccgccctg ggtgtttcta atgctgctat ggaggatttg ttaacagctgcaaccacggg 3420 cattttgagg cacattgcag ctgaagaagt gtctaaggaa agagagcgaagggagcagga 3480 gaggcagcgg gctgaagagg aaaggttgaa acaagagaga gagctggtgttaagtgagct 3540 gagccagggc ctggccgtgg agctgatgga acgcgtgatg atggagtttgtgagggaaac 3600 ctgctcccag gagttgaaga atgcagtaga gacagaccag agggtccgtgtggcccgttg 3660 ctgtgaggat gtctgtgccc acttagtgga cttgtttctc gtggaggaaatcttccagac 3720 tgcaaaggag accctccagg agcttcagtg cttctgcaag tatctacagcggtggaggga 3780 agctgtcaca gcccgcaaga aactgaggcg ccaaatgcgg gctttccctgctgcgccctg 3840 ctgcgtggac gtgagcgacc ggctgagggc gctggcgccc agcgcagagtgccccattgc 3900 tgaagagaac ctggccaggg gcctcctgga cctgggccat gcagggagattgggcatctc 3960 ttgcaccagg ttaaggcggc tcagaaacaa gacagctcac cagatgaaggttcagcactt 4020 ctaccagcag ctgctgagtg atgtggcatg ggcgtctctg gacctgccatccctcgtggc 4080 tgagcacctc cctgggaggc aggagcatgt gttttggaag ctggtgctggtgttgccgga 4140 tgtagaggag cagtccccag agagttgtgg cagaattcta gcaaattggttaaaagtcaa 4200 gttcatggga gatgaaggct cagtggatga cacatccagc gatgctggtgggattcagac 4260 gctttcgctt ttcaactcac ttagcagcaa aggggatcag atgatttctgttaacgtgtg 4320 tataaaggtg gcccatggcg ccctcagtga tggtgccatt gatgctgtggagacacagaa 4380 ggacctcctg ggagccagtg ggctcatgct gctgcttccc cccaaaatgaagagtgagga 4440 catggcagag gaggacgtgt actggctgtc ggccttgctg cagctcaagcagctcctgca 4500 ggctaagccc ttccagcctg cgcttcctct ggtggttctt gtgcctagcccaggagggga 4560 cgccgttgag aaggaagtag aagatggtct gatgctacag gacttggtttcagctaagct 4620 gatttcagat tacactgtta ccgagatccc tgataccatt aatgatctacaaggttcaac 4680 taaggttttg caagcagtgc agtggctggt ttcccactgc ccccattcccttgacctctg 4740 ctgccagact ctcattcagt acgtcgaaga cgggattggc catgagtttagtggccgctt 4800 tttccatgac agaagagaga ggcgtctggg cggtcttgct tctcaggagcctggcgccat 4860 cattgagctg tttaacagtg tgctgcagtt cctggcttct gtggtgtcctctgaacagct 4920 gtgtgacctg tcctggcctg tcactgagtt tgctgaggca gggggcagccggctgcttcc 4980 tcacctgcac tggaatgccc cagagcacct ggcctggctg aagcaggctgtgctcgggtt 5040 ccagcttccg cagatggacc ttccacccct gggggccccc tggctccccgtgtgctccat 5100 ggttgtccag tacgcctccc agatccccag ctcacgccag acacagcctgtcctccagtc 5160 ccaggtggag aacctgctcc acagaaccta ctgtaggtgg aagagcaagagtccctcccc 5220 agtccatggg gcaggcccct cggtcatgga gatcccatgg gatgatcttatcgccttgtg 5280 tatcaaccac aagctgagag actggacgcc cccccggctt cctgttacatcagaggcgct 5340 gagtgaagat ggtcagatat gtgtgtattt ttttaaaaac gatttgaaaaaatatgatgt 5400 tcctttgtcg tgggaacaag ccaggttgca gacgcagaag gagctacagctgagagaggg 5460 acgtttggca ataaagcctt ttcatccttc tgcaaacaat tttcccataccattgcttca 5520 catgcaccgt aactggaaga ggagcacaga gtgtgctcaa gaggggaggattcccagcac 5580 agaggatctg atgcgaggag cttctgctga ggagctcttg gcgcagtgtttgtcgagcag 5640 tctgctgctg gagaaagaag agaacaagag gtttgaagat cagcttcagcaatggttgtc 5700 tgaagactca ggagcattta cggatttaac ttcccttccc ctctatcttcctcagactct 5760 agtgtctctt tctcacacta ttgaacctgt gatgaaaaca tctgtaactactagcccaca 5820 gagtgacatg atgagggagc aactgcagct gtcagaggcg acaggaacgtgtctaggcga 5880 acgactaaag cacctggaaa ggctgatccg gagttcaagg gaagaggaagttgcctctga 5940 gctccatctc tctgcgctgc tagacatggt ggacatttga gcagcctgacctgtggggag 6000 ggggtctctc ccgaagagtt tctgttttta ctcaaaataa tgttattctcagatgcttga 6060 tgcactgttg gaaatgtgat taatttaatc atgcagataa accatttaaatgtc 6114 5 20 DNA Artificial Sequence Forward Primer 5 ccgtgggatgacatcatcac 20 6 20 DNA Artificial Sequence Reverse Primer 6 catgtccaccatctccagca 20 7 20 DNA Artificial Sequence Primer 7 tttgtctggaggatgatcgc 20 8 20 DNA Artificial Sequence Primer 8 aaagagaaaggggccaggcc 20 9 20 DNA Artificial Sequence Primer 9 ccagcttcttgtccaaaagc 20

What is claimed is:
 1. An isolated protein having the amino acidsequence shown in SEQ ID No.
 1. 2. A variant protein having a kinaseactivity substantially similar to the protein recited in claim 1,wherein 1-20 amino acids are deleted, substituted, and/or added.